Monograph Series No. 19 - Uterine Infections in Mares and Women - A Comparative Study II

Proceedings of a Workshop on

UTERINE INFECTION IN MARES AND WOMEN: A COMPARATIVESTUDY II

9th – 13th November 2005South Carolina, USA

Editors: M. M. LeBlanc, J. F. Wade and L. Foster

Havemeyer Foundation

Havemeyer Foundation Monograph Series No. 19

© 2008 by R & W CommunicationsSuites 3 & 4, 8 Kings Court, Willie Snaith Road, Newmarket, Suffolk CB8 7SG, UK

No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means,electronic, mechanical, photocopying, recording or otherwise, without the prior permission of the copyright owner.Authorisation to photocopy items for internal or personal use, or the internal or personal use of specific clients, isgranted by R & W Communications for libraries and other users registered with the Copyright Clearance Center (CCC)Transactional Reporting Service, provided that the base fee of £0.02 per copy (no additional fee per page) is paiddirectly to CCC, 21 Congress Street, Salem, MA 01970. This consent does not extend to other kinds of copying, suchas copying for general distribution, for advertising or promotional purposes, for creating new collective works, or forresale.

First published 2008

ISSN 1472-3158

Published by R & W Communications

Printed in Great Britain by Quality Print Services (Anglia) Limited

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CONTENTS

FOREWORD ......................................................................................................................................Page v

SESSION 1: REVIEW OF THE PROBLEM

Reproductive physiology of the late gestation mareD. Paccamonti ...................................................................................................................................Page 3

The equine placenta: infections of the gravid uterus from a placental perspectiveD. H. Schlafer ....................................................................................................................................Page 6

Neonatal diseases of foalsJ. E. Palmer .......................................................................................................................................Page 7

SESSION 2: ANIMAL MODELS

Animal models of pre-term birthM. A. Elovitz.....................................................................................................................................Page 11

Findings of a model of chronic ascending placentitis in the mareM. M. LeBlanc, G. D. Lester, P. J. Hansen, J. A. McGlothlin, R. J. Stawicki and D. Paccamonti .................................................................................................................................Page 12

Experimentally-induced placentitis in late gestation ewes and mares: evaluation of pathogenprogression using Lux-modified bacteria and bioluminescence imagingP. Ryan, K. Moulton, S. Willard, J. Crouch, D. Sykes, D. Christiansen and W. Bennett................Page 13

Allantoic catheterisation of the pregnant mare: development of the technique andpreliminary findingsS. K. Lyle, D. Paccamonti, J. D. Hubert, J. R. Johnson, R. C. Causey and B. E. Eilts..................Page 15

Trophoblast cell phagocytosis: an in vivo study of early events in establishment of equinebacterial placentitisT. Infernuso, S. Fubini, J. Cheetham, H. Erb, D. Volkmann, R. Gleed and D. H. Schlafer ...........Page 16

Identification of novel biomarkers of intra-amniotic infection by proteomic profilingM. G. Gravett...................................................................................................................................Page 17

Molecular pathogenesis of the genital mycoplasmasM. Peltier .........................................................................................................................................Page 18

The use of betamethasone to advance fetal maturation in the equineD. Christiansen, G. Olsen, J. Smith, R. Hopper, M. M. LeBlanc and P. Ryan ...............................Page 19

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Uterine Infection in Mares & Women: A Comparative Study II

SESSION 3: PATHOLOGICAL FINDINGS

Equine placental pathology: the common and the not so commonN. M. Williams, J. M. Donahue, D. C. Bolin, R. C. Giles, L. R. Harrison, C. B. Hong,C. B. Jackson, K. B. Poonacha and M. L. Vickers..........................................................................Page 23

Gross and microscopic pathology of placentas and foal organs in experimentally induced equine ascending bacterial placentitisM. B. Calderwood Mays, M. M. LeBlanc and M. L. Macpherson..................................................Page 27

SESSION 4: TREATMENT STRATEGIES FOR INFECTION IN LATE GESTATION

Innate immunity and microbiologically informed approaches to prevention and treatment of intrauterine infection during pregnancyJ. A. McGregor, O. Equils, M. Wilson and S. Witkin ......................................................................Page 31

Novel treatment strategies for infection-induced pre-term birth: a non-human primate modelM. G. Gravett...................................................................................................................................Page 32

Detection of gentamicin and penicillin concentrations in allantoic fluid of pregnant pony maresby in vivo microdialysisT. A. Murchie, M. L. Macpherson, M. M. LeBlanc, S. Luznar and T. W. Vickroy ..........................Page 33

Placental penetration of trimethoprim sulfamethoxazole and pentoxifylline in mares with placentitisS. Rebello, M. L. Macpherson, T. Murchie, M. M. LeBlanc and T. W. Vickroy ..............................Page 34

Experimentally induced placentitis in late gestation mares with Streptococcus equi zooepidemicus:prevention of pre-term birthP. Ryan, J. Crouch, D. Sykes, K. Moulton, D. Christiansen, R. Hopper, R. ReadW. Bennett and M. M. LeBlanc........................................................................................................Page 35

Progestins and pre-term birth: what is the evidence and does it make sense?M. A. Elovitz ....................................................................................................................................Page 37

The role of endogenous and exogenous progestagens in pregnant maresJ. C. Ousey, P. D. Rossdale, E. Houghton, L. Grainger, L. Palmer and A. L. Fowden..................Page 38

Omega-3 (n-3) fatty acid nutrition: optimising reproductive and early life neurologicalperinatal outcomesJ. A. McGregor and M. Harris........................................................................................................Page 42

SESSION 5: EFFECTS OF THE NEONATE

Placentitis associated neonatal problems and the effect of therapeutic interventionsJ. E. Palmer, P. L. Sertich, B. I. Cornman, P. A. Wilkins and R.C. Boston.....................................Page 45

Evaluation of coagulation in neonatal foals born to mares with and without placentitisA. Bentz, P. L. Sertich, R. C. Boston, P. A. Wilkins and J. E. Palmer.............................................Page 46

Equine neonatal sepsis: short and long-term outcomesL. C. Sanchez and G. D. Lester .......................................................................................................Page 48

The outcome of foals born to mares treated for placentitisB. S. Barr .........................................................................................................................................Page 49

APPENDIX

Ascending placentitis: what we know about pathophysiology, diagnosis and treatment M. M. LeBlanc, M. Macpherson and P. Sheerin .............................................................................Page 53

LIST OF PARTICIPANTS..................................................................................................................Page 71

AUTHOR INDEX ..............................................................................................................................Page 72

FOREWORD

Being given the opportunity to host a

Havemeyer Foundation Workshop is an

honor and a challenge. It is an honor

because one is given funds to design a scientific

workshop in a venue of their choosing and invite

prominent scientists and their graduate students to

discuss a significant problem in the horse industry

for three days. It is a challenge because

participation is limited to 30 individuals in an

attempt to foster animated discussion and

collaborations. The second workshop on infections

in late gestation: comparisons between women and

horses brought old and new faces to Hilton Head

South Carolina for three sunny beautiful days in

November 2005. It was a huge success in regards to

science, camaraderie and the formation of new

collaborations. The group included three human

perinatologists and a basic scientist who gladly

shared their clinical expertise in human medicine

and their scientific acumen on animal models with

veterinary clinicians, reproductive physiologists

and pathologists. Members of three veterinary

colleges presented work from their models of

placentitis while equine neonatologists discussed

neonatal outcome. And our reproductive

pathologists reminded us how infection gravely

affects reproductive biology and neonatal outcome.

Discussions far outlasted the time allotted them.

Three topics that will require future study were

outlined on the last day.

• There is a need for uniformity in the models of

equine placentitis. There are currently three

models of infection in the USA. Experimental

numbers are small in each model and there is

lack of uniformity between models. It was

decided that the three groups would identify an

inoculum that would be used in all models to

enable future comparisons.

• Treatment studies in human medicine, laboratory

animal models and in the mare indicate that

pregnancy can be prolonged in the face of

infection with drugs (systemic antibiotics, non

steroidal anti-inflammatory drugs and

tocolytics), however, neonatal outcome is not

necessarily improved. Preliminary data on

administration of dexamethazone to the dam

during infection in laboratory models appears to

improve neonatal survival. Its use needs to be

investigated in the horse in an attempt to improve

neonatal outcome.

• The cervix is ‘sacred’. If it is damaged or loses

its seal, ascending infection can seed the fetal

membranes. Our knowledge of how the cervix

functions at the molecular level during

pregnancy or even during the estrous cycle is

limited. Nor do we have a clear understanding of

how hormonal interactions are changed during

infection.

The monograph that follows includes a review

article presented at the Annual Convention of the

American Association of Equine Practitioners

followed by long and short abstracts submitted by

participants. The length of the abstracts varies as

some work presented at the workshop has not been

published. Current restrictions on abstract length

have prohibited some investigators from including

extended abstracts.

As always Gene, thank you. The Foundation has

given me some of the most memorable days in my

life. I would have never dreamed that a prominent

human perinatologist would share similar concerns

over the fragility of the cervix in regards to

pregnancy maintenance until ‘A mighty fortress is

the cervix’ was sung to me at dinner and there was

more than one verse.Michelle LeBlanc

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HAVEMEYER SCIENTIFIC WORKSHOPS

1981 First International Workshop on Lymphocyte Alloantigens of the HorseOctober - New York City, USAOrganiser: Dr D. F. Antczak

1982 Second International Workshop on Lymphocyte Alloantigens of the HorseOctober - Cornell University, Ithaca, New York, USAOrganiser: Dr D. F. Antczak

1983 Third International Workshop on Lymphocyte Alloantigens of the HorseApril - New Bolton Center, University of Pennsylvania, USAOrganiser: Dr D. F. Antczak

1984 First International Symposium on Equine Embryo TransferOctober - Cornell University, Ithaca, New York, USAOrganisers: Drs D. F. Antczak and W. R. Allen

1985 Fourth International Workshop on Lymphocyte Alloantigens of the HorseOctober - University of Kentucky, USAOrganisers: Drs D. F. Antczak and E. Bailey

1986 Workshop on Corynebacterium equi Pneumonia of FoalsJuly - University of Guelph, CanadaOrganiser: Dr J. F. Prescott

1987 Fifth International Workshop on Lymphocyte Alloantigens of the HorseOctober - Louisiana State University, USAOrganisers: Drs D. F. Antczak and J. McClure

1989 Second International Symposium on Equine Embryo TransferFebruary - Banff, Alberta, CanadaOrganisers: Drs D. F. Antczak and W. R. Allen

1990 International Workshop on Equine SarcoidsApril - Interlaken, SwitzerlandOrganisers: Dr D. F. Antczak and Professor S. Lazary

1992 Workshop on Equine Neonatal MedicineJanuary - Naples, FloridaOrganisers: Drs D. F. Antczak and P. D. Rossdale

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Third International Symposium on Equine Embryo TransferFebruary - Buenos Aires, Argentina

Organisers: Drs D. F. Antczak, W. R. Allen, J. G. Oriol and R. Pashen

1995 Equine PerinatologyJuly - Cambridge, England

Organiser: Dr P. D. Rossdale

Second International Equine Leucocyte Antigen WorkshopJuly - Lake Tahoe, California, USA

Organisers: Drs D. F. Antczak, P. Lunn and M. Holmes

First International Workshop on Equine Gene MappingOctober - Lexington, Kentucky, USA

Organisers: Drs D. F. Antczak and E. Bailey

Erection and Ejaculation in the Human Male and Stallion: A Comparative StudyOctober - Mount Joy, Pennsylvania, USA

Organiser: Dr S. M. McDonnell

Bone Remodelling WorkshopOctober - Corcord, Massachusetts, USA

Organiser: Dr H. Seeherman

1997 Second International Workshop on Equine Gene MappingOctober - San Diego, California, USA


Maternal Recognition of Pregnancy in the MareJanuary - Dominican Republic

Organisers: Drs W. R. Allen and T. A. E. Stout

Uterine ClearanceMarch - Gainesville, Florida, USA

Organiser: Dr M. M. LeBlanc

Trophoblast DifferentiationSeptember - Edinburgh, Scotland

Organisers: Drs D. F. Antczak and F. Stewart

1998 Third International Genome WorkshopJanuary - San Diego, California, USA


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Third International Workshop on Perinatology: Genesis and Post Natal Consequences of Abnormal Intra-uterine Developments: Comparative AspectsFebruary - Sydney, AustraliaOrganiser: Dr P. D. Rossdale

Horse Genomics and the Genetic Factors Affecting Race Horse Performance March - Banbury Center, Cold Spring Harbor, New York, USAOrganisers: Drs D. F. Antczak, E. Bailey and J. Witkowski

Allergic Diseases of the HorseApril - Lipica, SloveniaOrganisers: Drs D. F. Antczak, S. Lazary and E. Marti

Equine Placentitis WorkshopOctober - Lexington, Kentucky, USAOrganisers: Drs D. F. Antczak, W. R. Allen and W. Zent

Septicemia II WorkshopNovember - Boston, Massachusetts, USAOrganiser: Dr M. R. Paradis

1999 Equine Genome ProjectJanuary - San Diego, California, USAOrganisers: Drs D. F. Antczak and E. Bailey

Third International Equine Genome WorkshopJune - Uppsala, SwedenOrganisers: Drs D. F. Antczak, E. Bailey and K. Sandberg

Fourth International Meeting of OIE and WHO Experts on Control of Equine InfluenzaAugust - Miami, Florida, USAOrganiser: Dr J. Mumford

European Equine Gamete WorkshopSeptember - Lopuszna, PolandOrganisers: Drs W. R. Allen and M. Tischner

Fetomaternal Control of PregnancyNovember - Barbados, West IndiesOrganisers: Drs T. Stout and W. R. Allen

2000 Equine Genome ProjectJanuary - San Diego, California, USAOrganisers: Drs D. F. Antczak and E. Bailey

Uterine Infections in Mares and Women: A Comparative StudyMarch - Naples, Florida, USAOrganiser: Dr M. M. LeBlanc

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5th International Symposium on Equine Embryo TransferJuly - Saari, FinlandOrganiser: Dr T. Katila

2001 USDA International Plant & Animal Genome ConferenceJanuary - San Diego, California

Equine Immunology in 2001January - Santa Fe, New MexicoOrganiser: Dr D. P. Lunn

Asthma and Allergies II April - HungaryOrganisers: S. Lazary and E. Marti

From Elephants to Aids June - Port Douglas, AustraliaOrganiser: Professor W. R. Allen

International Equine Gene Mapping July - Brisbane, AustraliaOrganiser: K. Bell

Second Meeting of the European Gamete Group (EEGG) September - Loosdrecht, The NetherlandsOrganiser: Dr T. A. E. Stout

Foal Septicemia IIIOctober - Tufts University European Center, Talloires, FranceOrganiser: M. R. Paradis

Infectious Disease Programme for the Equine Industry and VeterinaryPractitionersOctober - Marilyn duPont Scott Medical Center, Morvan Park, Virginia, USAOrganisers: Drs J. A. Mumford and F. Fregin

From Epididymis to EmbryoOctober - Fairmont Hotel, New Orleans, USAOrganiser: Dr L. H-A. Morris

2002 USDA International Plant & Animal Genome ConferenceJanuary - San Diego, California

Comparative Neonatology/PerinatologyMarch - Palm Springs, CaliforniaOrganiser: P. Sibbons

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Stallion Behaviour IVJune - Reykjavik, IcelandOrganisers: S. McDonell and D. Miller

Rhodococcus Equi IIJuly - Pullman, WashingtonOrganiser: J. Prescott

Equine Orthopaedic InfectionAugust - Dublin, IrelandOrganiser: E. Santschi

Inflammatory Airway DiseaseSeptember - Boston, USAOrganiser: Dr E. Robinson

2003 USDA International Plant and Animal Genome ConferenceJanuary - San Diego, California

Embryonic and Fetal NutritionMay - Ravello, ItalyOrganiser: S. Wilsher

Genomics and the Equine Immunity SystemJune - Ithaca, New YorkOrganiser: D. F. Antczak

Fifth International Gene Mapping WorkshopAugust - Kreuger Park, South AfricaOrganiser: E. Baily and E. Vandyke

Equine Recurrent Laryngeal NeuropathySeptember - Stratford-upon-Avon, UKOrganisers: P. Dixon and E. Robinson

Transporting Gametes and EmbryosOctober - Brewster, MassachusettsOrganiser: E. Squires

Third Meeting of the European Gamete Group (EEGG) October - Pardubice, Czech RepublicOrganiser: J. and Z. Müller

Nosocomial Infections and Biosecurity in Equine HospitalsOctober - Lexington, USAOrganiser: F. Bain and J. Taub-Dargatz

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2004 USDA International Plant and Animal Genome ConferenceJanuary - San Diego, California

Equine Viral Herpesvirus WorkshopJune/July - Tuscany, ItalyOrganiser: P. Lunn

Equine Embryo Transfer VI WorkshopAugust - Rio de Janiero, BrazilOrganiser: M. Alvarenga

Sporting Injuries in Horses and Man: A Comparative ApproachSeptember - Lexington, USAOrganiser: E. J. L. Soulsby

Maternal Recognition of Pregnancy in the Mare IIINovember - Barbados, West IndiesOrganiser: T. A. E. Stout

2005 USDA International Plant and Animal Genome ConferenceJanuary - San Diego, CaliforniaOrganiser: J. Mickelson

Comparative PlacentologyApril - Victoria, CanadaOrganiser: P. Sibbons

Sixth International Gene MappingJuly - Dublin, IrelandOrganisers: E. Bailey and J. Flynn

World Equine Airway SymposiumJuly - Ithaca, USAOrganisers: D. Ainsworth, E. Robinson, N. DuCharme, B. McGorum and L. Viel

Genetic Relatedness Between Different Breeds of Horses using MolecularMarkersAugust - PolandOrganisers: M. Binns, G. Lothran and B. Graiak

International Equine Gamete GroupSeptember - Kühlungsborn, GermanyOrganisers: H. Alm, H. Torner, K. Hinrichs and E. Squires

Biomarkers for Diagnosing Musculoskeletal DiseaseOctober/November - Colorado, USAOrganiser: W. McIllwraith

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Equine Influenza and Cross Species TransmissionNovember - Florida, USAOrganiser: J. Mumford

Uterine Infection in Mares & Women: A Comparative Study IINovember - South Carolina, USAOrganiser: M. M. LeBlanc

2006 Exercise-Induced Pulmonary Haemorrhage: State of Current KnowledgeMarch - Vancouver, CanadaOrganisers: D.J. Marlin and K.W. Hinchcliff

HAVEMEYER MONOGRAPH SERIES

The following are monographs available to date at a cost of £9.95 each.

Series No 1PROCEEDINGS OF THE FIRST MEETING OF THE EUROPEAN EQUINE GAMETE GROUP (EEGG)Editors: W. R. Allen and J. F. Wade5th–8th September 1999 Lopuszna, Poland

Series No 2PROCEEDINGS OF A WORKSHOP ON FETOMATERNAL CONTROL OF PREGNANCY

Editors: T. A. E. Stout and J. F. Wade14th–16th November 1999Barbados, West Indies

Series No 3PROCEEDINGS OF THE 5TH INTERNATIONAL SYMPOSIUM ON EQUINE EMBRYO TRANSFER

Editors: T. Katila and J. F. Wade6th–9th July 2000Saari, Finland

Series No 4PROCEEDINGS OF A WORKSHOP ON EQUINE IMMUNOLOGY IN 2001 Editors: D. P. Lunn and J. F. Wade24th–28th January 2001Santa Fe, New Mexico

Series No 5PROCEEDINGS OF THE SECOND MEETING OF THE EUROPEAN GAMETE GROUP (EEGG) Editors: T. A. E. Stout and J. F. Wade26th–29th September 2001Loosdrecht, The Netherlands

Series No 6PROCEEDINGS OF A WORKSHOP ENTITLED FROM EPIDIDYMIS TO EMBRYO

Editors: L. H-A. Morris, L. Foster and J. F. Wade18th–21st October 2001New Orleans, USA

Series No 7FOURTH INTERNATIONAL MEETING OF OIE AND WHO ON CONTROL OF EQUINE INFLUENZA

Editors: J. A. Mumford and J. F. Wade3rd–5th August 1999Crowne Plaza Hotel, Miami, Florida, USA

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Series No 8PROCEEDINGS OF A WORKSHOP ON COMPARATIVE NEONATOLOGY/PERINATOLOGY

Editors: Dr P. Sibbons, L. Foster and J. F. Wade13th–15th March 2002Palm Springs, California, USA

Series No 9PROCEEDINGS OF A WORKSHOP ON INFLAMMATORY AIRWAY DISEASE:DEFINING THE SYNDROME

Editors: A. Hoffman, N. E. Robinson and J. F. Wade30th September–3rd October 2002Boston, USA

Series No 10PROCEEDINGS OF A WORKSHOP ON EMBRYONIC AND FETAL NUTRITION

Editors: S. Wilsher and J. F. Wade15th–18th May 2003Ravello, Italy

Series No 11PROCEEDINGS OF A WORKSHOP ON EQUINE RECURRENT LARYNGEAL NEUROPATHY

Editors: P. Dixon, E. Robinson and J. F. Wade7th–10th September 2003Stratford-upon-Avon, UK

Series No 12PROCEEDINGS OF A WORKSHOP ON TRANSPORTING GAMETES AND EMBRYOS

Editors: E. Squires and J. F. Wade2nd–5th October 2003Brewster, Massachusetts, USA

Series No 13PROCEEDINGS OF THE THIRD MEETING OF THE EUROPEAN GAMETE GROUP (EEGG) Editors: J. Müller, Z. Müller and J. F. Wade12th–15th October 2003Pardubice, Czech Republic

Series No 14PROCEEDINGS OF THE 6TH INTERNATIONAL SYMPOSIUM ON EQUINE EMBRYO TRANSFER

Editors: M. Alvarenga and J. F. Wade4th–6th August 2004Rio de Janerio, Brazil

Series No 15PROCEEDINGS OF A WORKSHOP ON SPORTING INJURIES IN HORSES AND MAN:A COMPARATIVE APPROACH

Editors: Lord Soulsby and J. F. Wade23rd–25th September 2004Lexington, USA


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Series No 16PROCEEDINGS OF A WORKSHOP ON MATERNAL RECOGNITION OF PREGNANCY IN THE MARE III Editors: T. A. E. Stout and J. F. Wade13th–16th November 2004Barbados, West Indies

Series No 17PROCEEDINGS OF A WORKSHOP ON COMPARATIVE PLACENTOLOGY

Editors: P. Sibbons and J. F. Wade21st–24th April 2005Victoria, Canada

Series No 18PROCEEDINGS OF THE FIRST INTERNATIONAL EQUINE GAMETES GROUP WORKSHOP

Editors: A. Hannelore, H. Torner, K. Hinrichs, E. Squires and J. F. Wade18th–21st September 2005Rostock, Germany

If you wish to order copies, please contact R & W Communications, Suites 3 & 4, 8 Kings Court, WillieSnaith Road, Newmarket, Suffolk CB8 7SG, UK, Tel: +44 1638 667600, Fax: +44 1638 667229, e-mail: [email protected]


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1

SESSION 1:

Review of the problem

Chairman: M. M. LeBlanc

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REPRODUCTIVE PHYSIOLOGY OF THE LATEGESTATION MARE

D. Paccamonti

Department of Veterinary Clinical Sciences, Louisiana State University, Baton Rouge, Louisiana 70803 USA

The scope of this paper is not to review thoroughlyour knowledge of the physiology of the lategestation mare. Rather it is a brief overview ofsome aspects of late gestation in the mare with anattempt to highlight some of the similarities anddifferences between mares and women. The goalin horse breeding is to raise an athlete, whether abody builder (ie halter class Quarter Horse) ortrack star (ie Thoroughbred). Successfultherapeutic options for the compromised fetus orneonate must achieve this goal. In the case of apremature human infant, an economic value is notassigned and no financial limit is put on treatmentoptions. However, with a foal, economics areoften a limiting factor and mere survival is notusually considered a satisfactory outcome.

The human neonatal Intensive Care Unit(ICU) model for treatment of prematurelydelivered infants may not serve the equineindustry as well. Because of the advances inhuman neonatal medicine, infants can be deliveredevermore prematurely, with a good chance ofsurvival. Nevertheless, it is well known that intra-uterine growth retardation and premature deliverymay have ramifications much later in life (Barkeret al. 1989). For example, it has been reported thatlow birth weight resulting from intra-uterinegrowth restriction was associated with decreasedbrain weight and increased adiposity at maturity insheep (Louey et al. 2005). How the effects of pre-term delivery might manifest themselves in theequine adult is unknown.

While great strides have been made in humanneonatal intensive care, enabling earlier and earlierdelivery of premature infants, the same cannot besaid for equine neonatology. The ability to deliveran at-risk human fetus, provide treatment and carefor it outside the womb is a luxury that is not anoption in veterinary medicine. We are often unable

to support a severely compromised premature foaloutside the mare’s uterus, and we lack a clear timepoint in gestation after which we can expect afavourable outcome to treatment. Because of thepoor prognosis for survival after delivery of apremature foal, treatment directed at maintainingthe pregnancy or stimulating precociousmaturation rather than inducing delivery currentlyoffers the best hope for success. If an equineneonate can be maintained in utero for a time afterdiagnosis of placental disease, precociousdevelopment may occur and the foal can be bornearly, seemingly mature and with a good chance ofsurvival, after a gestation period usuallyconsidered too short for normal fetal maturity.

Understanding the pathogenesis of pre-termdelivery is critical in developing effective treatmentmodalities. Should treatment be aimed at directlyinhibiting myometrial activity, preventing the switchfrom contractures to contractions; or at controllingpossible infection and the associated inflammatoryprocess, thereby promoting myometrial quiescence?The current working hypothesis of pre-term deliverydue to placentitis attributes fetal loss to a rise inprostaglandins resulting from an increase in pro-inflammatory cytokines (LeBlanc et al. 2004). Ifthis hypothesis is supported by future studies,treatment modalities must be directed at controllingthe bacterial infection, preventing the rise in pro-inflammatory cytokines and prostaglandins andinhibiting myometrial contractions (LeBlanc et al.2004). In support of this concept, pre-term deliveryin sheep initiated with RU486 was successfullyprevented by the PGF2α receptor antagonistTHG113.31 (Hirst et al. 2005).

Typical therapy for placentitis includessystemic antibiotics, most commonly trimethoprimsulfa because of its oral availability. Pentoxyfillineis often administered because of its anti-

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inflammatory properties and ability to improvetissue perfusion. Studies examining concentrationsof antibiotics such as trimethoprim sulfa andpentoxyfilline in allantoic fluid have shown thatthese commonly used therapeutic drugs do achieveappreciable levels in the fetal compartment(LeBlanc et al. 2004). Exogenous progestogen,usually altrenogest at double the recommendeddose, is given to maintain myometrial quiescence.

Although treatment of the equine fetus inutero is usually via systemic treatment of the dam,direct injection into the allantoic cavity may be anoption to consider. A report by Stefos et al. (2005)described treatment of anhydramnios by serialinfusion of normal saline into the amniotic cavity.In the cases described, one woman received 3infusions and the other 4 infusions of 100 or 200ml saline at 4 or 5 week intervals. Both womengave birth to normal infants. In an earlier report,transcervical amnioinfusion of amphotericin wasused successfully to treat an intra-amnioticCandida albicans infection, resulting in the birthof a normal infant (Shalev et al. 1994).

Urinary trypsin inhibitor (UTI) is an anti-inflammatory substance that is also a potentinhibitor of myometrial contractions (Kanayamaet al. 1995a; Pugia and Lott 2005). Normallypresent in human amniotic fluid, UTI has beenreported to be capable of preventing pre-termdelivery induced by lipopolysaccharide (LPS)through suppression of cytokine production(Kaga et al. 1996). Urinary trypsin inhibitorinhibits in vitro production of PGE2 bymyometrial cells stimulated with IL-1 and LPS (elMaradny et al. 1996), suppressed IL-1, IL-6 andTNFα after LPS administration in mice (Kaga etal. 1996), and inhibited cervical softening anddilation induced by IL-8 in rabbits (Kanayama etal. 1995b). In Japan, UTI has been shown to beclinically effective in inhibiting uterinecontractions and has been used to preventpremature delivery in women (Kanayama et al.1996). Whether UTI plays a role in equinepregnancy, or may be of therapeutic value, isunknown at this time.

Various aspects of late gestation in the maremake horses unique among the domestic species.Gestation length is extremely variable, with therange for normal gestation length in horsesencompassing a 6-week period. In no otherspecies is the concept of ‘readiness for birth’ soapparent. Fetal maturation is important in timingthe onset of parturition in most species but

variability is expressed in days, not weeks.Increasing oestrogens and declining progestogensat the end of gestation are characteristic of manydomestic species. In horses, however, oestrogensdecline and progestogens rise in the last weeks ofgestation, followed by a rapid decline inprogestogens in the last days before delivery.Abnormal progestogen concentrations may signalplacental disease. A precocious rise inprogestogens is often observed with placentitis.Recently, Ousey et al. (2005) reported thatincreased progestogens were found in mares withplacentitis compared to mares with normalpregnancies, indicating increased fetal productionor increased utero-placental metabolism inresponse to the chronic stress. Stawicki et al.(2002) and Leblanc et al. (2004) reported similarfindings in a model of experimental placentitis.Conversely, an early decline in progestogens isusually associated with acute stress and abortion(LeBlanc et al. 2004). As with many otherspecies, fetal adrenocortical hormones increasenear term but, in the horse, this change onlyoccurs within a few days of parturition. The risein fetal cortisol is associated with a number ofchanges associated with fetal maturation andreadiness for birth, such as an increase in thyroidhormone, an increase in the neutrophil:lymphocyte ratio, and maturation of the fetal lungand gut. Foals delivered pre-term with precociouschanges in progestogens often have concurrentprecocious adrenocortical activity and haveimproved chances of survival.

In comparative studies of gestational diseasein mares and women, the presence of 2 fetalcompartments (amniotic and allantoic) in themare, in comparison to a single compartment inhumans, is an important difference. The allantoicand amniotic compartments are dissimilar inorigin, components and dynamics, with many ofthe constituents changing over time. Amnioticfluid is derived primarily from the respiratory andgastrointestinal systems. Allantoic fluid is in largepart fetal urine; however, because of the fusion ofthe allantois with the chorion, and its relationshipto the maternal endometrium, changes in theconstituents of allantoic fluid may reflectplacental function. How do the allantoic andamniotic compartments behave in relation to fetalhealth and placental function? Often viewed as awaste receptacle, should the allantoiccompartment be viewed as more of a dynamiccomponent in fetal wellbeing? Further studies of

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the fluids during normal and abnormal gestationsmay shed light on the pathophysiology of diseaseand may lead to more successful treatmentmodalities.

REFERENCES

Barker, D.J., Winter, P.D., Osmond, C., Margetts, B. andSimmonds, S.J. (1989) Weight in infancy and deathfrom ischaemic heart disease. Lancet 2, 577-580.

el Maradny, E., Kanayama, N., Halim, A., Maehara, K.,Kobayashi, T. and Terao, T. (1996) Effects ofurinary trypsin inhibitor on myometrial contractionin term and pre-term deliveries. Gynecol. Obstet.Invest. 41, 96-102.

Hirst, J. J., Parkington, H. C., Young, I. R., Palliser, H.K., Peri, K. G. and Olson, D. M. (2005) Delay ofpre-term birth in sheep by THG113.31, aprostaglandin F-2 alpha receptor antagonist. Am. J.Obstet. Gynecol. 193, 256-266.

Kaga, N., Katsuki, Y., Futamura, Y., Obata, M. andShibutani, Y. (1996) Role of urinary trypsininhibitor in the maintenance of pregnancy in mice.Obstet. Gynecol. 88, 872-882.

Kanayama, N., el Maradny, E., Halim, A., Liping, S.,Maehara, K., Kajiwara, Y. and Terao, T. (1995a)Urinary trypsin inhibitor prevents uterine musclecontraction by inhibition of Ca{++} influx. Am. J.Obstet. Gynecol. 173, 192-199.

Kanayama, N., el Maradny, E., Halim, A., Maehara, K.,Kajiwara, Y. and Terao, T. (1995b) Urinary trypsininhibitor suppresses premature cervical ripening.Eur. J. Obstet. Gynecol. Reprod. Biol. 60, 181-186.

Kanayama, N., el Maradny, E., Yamamoto, N.,

Tokunaga, N., Maehara, K. and Terao, T. (1996)Urinary trypsin inhibitor: a new drug to treat pre-term labour: A comparative study with ritodrine.Eur. J. Obstet. Gynecol. Reprod. Biol. 67, 133-138.

LeBlanc, M.M., Macpherson, M. and Sheerin, P. (2004)Ascending placentitis: What we know aboutpathophysiology, diagnosis, and treatment. Proc.Am. Assoc. Eq. Pract. 50, 127-143.

Louey, S., Cock, M. L. and Harding, R. (2005) Longterm consequences of low birthweight on postnatalgrowth, adiposity and brain weight at maturity insheep. J. Reprod. Dev. 51, 59-68.

Ousey, J.C., Houghton, E., Grainger, L., Rossdale, P.D.and Fowden, A.L. (2005) Progestagen profilesduring the last trimester of gestation inThoroughbred mares with normal or compromisedpregnancies. Theriogenology 63, 1844-1856.

Pugia, M.J. and Lott, J.A. (2005) Pathophysiology anddiagnostic value of urinary trypsin inhibitors. Clin.Chem. Lab. Med. 43, 1-16.

Shalev, E., Battino, S., Romano, S., Blondhaim, O. andBen-Ami, M. (1994) Intraamniotic infection withCandida albicans successfully treated withtranscervical amnioinfusion of amphotericin. Am. J.Obstet. Gynecol. 170, 1271-1272.

Stawicki, R.J., Ruebel, H., Hansen, P.J., Sheerin, B.R.,ODonnell, L.J., Lester, G.D., Paccamonti, D.L. andLeBlanc, M. (2002) Endocrinological findings in anexperimental model of ascending placentitis in themare. Theriogenology 58, 849-852.

Stefos, T., Staikos, I., Plachouras, N. and Dousias, V.(2005) Serial saline amnioinfusion from 16th weekof gestation resulted in successful outcome ofpregnancy: Report of two cases. Eur. J. Obstet. Gyn.Reprod. Biol. 122, 250-251.

6


THE EQUINE PLACENTA: INFECTIONS OF THEGRAVID UTERUS FROM A PLACENTALPERSPECTIVE

D. H. Schlafer

College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA

Uterine infections are common in both pregnantmares and women. Microbial invaders gain accessto the outer fetal membranes by either traversingthe cervical canal (‘ascending’ route), or bycrossing from the endometrium. Data from largeretrospective studies, done both in Europe and theUS reveal that placentitis is found commonly inaborting mares. Placentitis was present in 9.8% of1,252 equine abortions in the United Kingdom(Smith 2003) and placentitis was detected in24.7% of placentas examined from aborted,stillborn or premature foals (236 of 954 placentas)in a study from Kentucky (Hong 1993).

Ascending bacterial infections typicallyproduce visibly prominent, severe, subacute tochronic locally extensive necro-suppurativeplacentitis involving the chorioallantois adjacentto the inner os of the mare’s cervix. This area ofthe choriallantois is known as ‘the cervical star’.Bacteria most commonly responsible forascending infection in the mare includeStreptococcus zooepidemicus, Escherichia coli,Pseudomonas spp., etc. In marked contrast,placentitis that developes by extension of infectionfrom the endometrium typically produces a‘diffuse’ lesion pattern, which, when found,suggests either Leptospira spp. bacterial orCandida yeast infection. A third and very unusual,(and hence ‘diagnostic’) pattern of placentalinfection, is produced by ‘nocardioform’(Amycolaptosis, spp., Crosiella eq. sp. nov.)infections resulting in locally extensive chronicplacentitis which typically is limited to areas ofchorioallantois that occupy the base of the hornsand cranial-ventral uterine body.

Common features of inflamed chorioallantoisdetected by histopathology include: oedema,vascular congestion, tissue necrosis, inflammatorycell exudation, neovascularisation, fibrosis, and

reactive hyperplasia of the allantoic epithelium.Severe infections can result in locally extensiveareas of chorioallantoic infarction. Acute bacterialinfections induce neutrophilic cell infiltrationwhile more chronic infections are associated withmacrophage and, less commonly, lymphocyticinflammation.

Most placental infections in mares aresubacute to chronic, but inflamed and necrotictissues quickly release cytokines and othermediators that have broad-ranging effects.Placental separation, compromise of vascularperfusion and decrease of the exchange areaquickly combine to depress placental function.Expanded use of ultrasonography has enabledveterinary clinicians to routinely diagnose andtreat placentitis in their equine patients. Excitingnew opportunities exist for development ofinnovative methods and new model systems forstudy of the pathogenesis of equine bacterialplacentitis, such as those provided by use ofchronically instrumented mares and their fetuses.Better understanding of the pathogenesis ofbacterial placentitis and its associated fetalcompromise will provide guidance for thedevelopment of more rapid and accuratediagnostic tests, and clinical detection andtreatment regimes.

REFERENCES

Hong, C.B., Donahue, J.M., Giles, R.C. Jr, Petrites-Murphy, M.B., Poonacha, K.B., Roberts, A.W.,Smith, B.J., Tramontin, R.R., Tuttle, P.A. andSwerczek, T.W. (1993) Etiology and pathology ofequine placentitis. J. Vet. Diagn Invest. 5, 56-63.

Smith, K.C., Blunden, A.S., Whitwell, K.E., Dunn, K.A.and Wales, A.D. (2003) A survey of equineabortion, stillbirth, and neonatal death in the UKfrom 1988 to 1997. Equine vet. J. 35, 496-501.

7


NEONATAL DISEASES OF FOALS

J. E. Palmer

Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University ofPennsylvania, Kennett Square, Pennsylvania, USA

Disruption of the intra-uterine environment willnot only result in fetal distress, but may be a majorcause of neonatal diseases recognised in foals.Conditions of neonatal foals which may be directlyassociated with placentitis include prematurity,intrauterine growth restriction (IURG) andprecocious maturation. Other conditions that maybe associated with placentitis include the complexof Neonatal Encephalopathy (NE), NeonatalNephropathy (NN), Neonatal Gastroenteropathy(NG) and maladaptation of other systems.

Prematurity in the foal has been defined asbirth before 320 days gestation. The term'dysmature' has been used to describe foals withphysical characteristics of prematurity in a foalborn after a gestation more than 320 days. Thesedefinitions are problematic because of the widevariation in normal gestational length in mares.This has lead to confusion in diagnosingprematurity, dysmaturity and IUGR. In theauthor’s opinion it is more helpful to defineprematurity based on the mare’s normalgestational length, use IUGR for foals small forgestational age (which are not constitutionallysmall) and avoid using the term dysmaturity.

There is a neonatal multisystem maladaptationsyndrome which includes neurological, renal,gastrointestinal and other system malfunctionswhich may be secondary to placental disease. Themost prominent signs are neurologicalabnormalities traditionally referred to as NeonatalMaladjustment Syndrome (NMS). Researchfindings in other species early in the 1990s ledmany equine neonatologists to speculate about ahypoxic ischaemic or asphyxial origin for thissyndrome. The term Hypoxic IschaemicEncephalopathy (HIE) largely replaced NMS. It isclear, however, that despite attractiveexperimental models showing many similarities,

often this disease syndrome occurs in the absenceof a detectable hypoxic ischaemic insult. Becauseof this the author prefers to use the terms, such asNeonatal Encephalopathy, that do not specify anaetiology or pathogenesis but only the organdysfunction and age group. Most recently therehas been speculation that inflammatory mediatorssecondary to placentitis may (possibly byinitiating a hypoxic ischaemic insult) beresponsible for the multiorgan dysfunction.

Foals suffering from NE may show changes inresponsiveness, muscle tone, behavior, evidenceof brain stem damage or seizure-like behaviour.Changes in responsiveness include hyperesthesia,hyper-responsiveness, hyperexcitability, hypo-responsiveness, periods of somnolence orunresponsiveness. Often the foals go through aperiod of increased responsiveness followed by aperiod of decreased responsiveness. Changes inmuscle tone include increased extensor tonus,hypotonia and other less common changes such asneurogenic myotonia or failure to protract frontlegs. Changes in behaviour are very common andinclude loss of suckle response, loss of tonguecurl, loss of tongue co-ordination, disorientationespecially relative to the udder, aimlesswandering, loss of affinity for the dam andabnormal vocalisation. Foals with NE commonlyhave changes in respiratory patterns with centraltachypnoea, apneusis, apnoea, cluster breathing,ataxic breathing, Cheyne-Stokes breathing orcentral hypercapnia. Other signs of brain stemdamage include loss of thermoregulatory control,generalised weakness, anisocoria, pupillarydilation, pinpoint pupils, central hypotension,decreased responsiveness, difficult to arouse, lossof consciousness, vestibular signs (circling, headtilt), facial nerve paresis and a variety of othersigns. Foals with NE have a wide variety of signs

8


and degrees of severity. More than 90% ofaffected foals are normal within 10 days.

Foals may also develop renal maladaptationreferred to as Neonatal Nephropathy (NN). Thereis a wide spectrum of disease seen in cases of NNincluding incomplete transition from fetal renalphysiology, water/sodium retention, mild tubulardysfunction (sodium wasting), abnormal excretionof drugs (eg high amikacin trough levels), acutetubular necrosis or decreased glomerular filtrationrate (GFR). Often the signs of dysfunction aresubtle and easily overlooked unless anticipated.Although almost always transient, on occasionsignificant acute damage may lead to chronic renaldisease. These foals often have a decreased GFRas reflected by a slow decrease birth creatinine ordecreased creatinine clearance, delayed waterexcretion with oedema formation and weight gainand slow response to fluid challenges.

Neonatal Gastroenteropathy (NG) can bemanifested by a wide spectrum of signs rangingfrom mild indigestion with dysmotility and enemadependence to moderate disease with ileus,diapedesis of blood into the lumen and mucosaloedema to severe disease with epithelial necrosis,intussusceptions, structures, haemorrhagicgastritis/enteritis/colitis, and pneumatosisintestinalis. Even mild forms predispose to sepsisand systemic inlammatory response system

(SIRS) with increased likelihood of translocationof bacteria. Like NN, often the signs ofdysfunction are subtle and easily overlookedunless anticipated. The most commonmanifestation is dysmotility with meconiumretention and lack of faecal passage for days(range 2–30 days). Despite faecal retention, animportant aspect is lack of discomfort. Classically,foals with dysmotility will not return enema fluidor strain associated with rectal distension.

Often, affected foals have the triad of NE, NNand NG. Other problems seen include metabolicmaladaptation, autonomic failure and othersystemic problems. Foals born from anenvironment of placentitis commonly have ageneralised inflammatory response as reflected bysystemic and haematological reactions. Theactivation of inflammatory and anti-inflammatorycascades may support precocious maturation ofmany body systems and may, in fact, impart someprotection from systemic infections.

Prematurity and IURG are easily confused.Clinically prematurity is marked by low birthweight, small frame, thin, poor muscledevelopment, periarticular laxity and generalflexibility. IUGR is marked by apparent cachexiaand disproportional growth. Either may be a directresult of placentitis and it is common for both to bepresent concurrently.

9


SESSION 2:

Animal models

Chairman: M. Macpherson

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11


ANIMAL MODELS OF PRE-TERM BIRTH

M. A. Elovitz

University of Pennsylvania, 1353 BRB II/III, 421 Curie Boulevard, Philadelphia 19104, USA

Pre-term birth continues to pose a significantclinical dilemma and contributes to both acuteand long-term neonatal morbidity. Despiteefforts, the incidence of pre-term birth has notdecreased, partly because of the lack ofunderstanding of the mechanisms that triggerparturition. Animal models are essentialresearch tools for investigating the pathways thatpromote pre-term parturition and for testingtherapeutic interventions. In the last decade, agrowing body of evidence demonstrates thatinfection or inflammation is a significantcontributor to pre-term birth. Consequently,

many investigators have created animal modelsthat reflect these findings. Current animalmodels of pre-term parturition include severalspecies, varying means of inducing aninflammatory or infectious state, and differentroutes of administration. Although each of thesemodels can advance our knowledge, it isimportant to understand their advantages,disadvantages and unique characteristics. Anunderstanding of such models will hopefullypromote continued research that will ultimatelylead to a decrease in pre-term birth and animprovement in neonatal outcome.

12


FINDINGS OF A MODEL OF CHRONIC ASCENDINGPLACENTITIS IN THE MARE

M. M. LeBlanc, G. D. Lester, P. J. Hansen*, J. A. McGlothlin, R. J. Stawicki andD. Paccamonti†

Departments of Large Animal Clinical Sciences; *Animal Sciences, University of Florida, Gainesville,Florida; †Department of Clinical Sciences, Louisiana State University, Baton Rouge, Louisiana, USA

A model of ascending placentitis was developed inthe mare to characterise myoelectrical andendocrine patterns in late gestation and determinehow ascending placentitis altered these patterns.

Thirty ponies were grouped into controls(n=8), experimentally infected (n=4),instrumented control mares (n=7) andinstrumented infected mares (n=11). Pony mareswere instrumented with myometrial electrodes andallantoic fluid catheters (n=4) 7–10 days beforeexperimental mares received an intra-cervicalinoculation with Streptococcus equi subspecieszooepidemicus (Day 285–293 of gestation).Myometrial electrical activity was analysed duringthe early morning, late morning and evening hoursuntil delivery. Blood and allantoic fluid werecollected to measure progestins and prostaglandinF2α and E2.

Three of 15 inoculated mares exhibited nooutward signs of infection. Plasma progestinseither rose (n=8) or fell after inoculation (n=7) ininoculated mares, whereas levels remained atbaseline in control mares until approximately 21days before delivery when they began a steadyrise. Progestin levels declined in all mares in the24 h preceding delivery. All inoculated maresdelivered prematurely, 5–27 days afterinoculation. Thirteen of 15 foals were aborted and2 were viable. Control mares foaled after dGA

320. Control mares exhibited 2 types ofmyometrial activity, low amplitude epochs ofactivity that lasted for one min or more (largespike bursts) and high amplitude epochs ofactivity that were less than 30 s (small spikebursts). The duration and number of large spikeclusters varied little as gestation progressedwhereas the number of small spike bursts began toincrease at night in the last 6 days of gestation andcontinued to increase at night until parturition.Mares with experimentally induced placentitis didnot exhibit a rise in small burst clusters in the lastweek of gestation as did control mares. Theyexhibited an increase in the duration and intensityof the large spike bursts in the 4 days precedingparturition. Concentrations of prostaglandins(PGE2) and (PGF2α) in allantoic fluid samplescollected within 48 h of abortion or delivery inmares with experimentally-induced placentitisrose significantly and were higher than that ofcontrol mares.

These findings indicate that ascendingplacentitis due to Streptococcus equi subspecieszooepidemicus is associated with a rise in allantoicfluid prostaglandins, premature delivery and insome cases, accelerated maturation of the foal.Myoelectrical activity pattern in infected maresdiffers from that of non-infected mares that deliverat term.

13


EXPERIMENTALLY-INDUCED PLACENTITIS IN LATEGESTATION EWES AND MARES: EVALUATION OFPATHOGEN PROGRESSION USING LUX-MODIFIEDBACTERIA AND BIOLUMINESCENCE IMAGING

P. Ryan*, K. Moulton, S. Willard, J. Crouch, D. Sykes, D. Christiansen*

and W. Bennett†

Department of Animal and Dairy Sciences and *Department of Pathobiology and PopulationMedicine, Mississippi State University, Mississippi State, Mississippi; †Department of Obstetrics and Gynecology, University of Mississippi Medical School, Jackson, Mississippi, USA

Placental infection due to opportunistic pathogenssuch as Escherichia coli (E. coli) is the single mostcommon cause of abortion, stillbirth and prematuredelivery in women and domestic animal species.The consequence of placentitis during latepregnancy increases the incidence of abortion, pre-term delivery or stillbirth. Moreover, increasingevidence demonstrates that pathogen progressionduring placentitis may involve invasion of fetaltissues, including the brain, leading to increasedpro-inflammatory cytokine expression resulting inonset of premature delivery and/or fetalneurological damage. Thus, the objectives of thispilot study were 1) to develop a model for pre-termdelivery using late term pregnant ewes; and 2) todetermine pathogen progression and invasion ofthe fetal environment by experimentally-induceduterine infection with a lux gene-modified E. coliusing real time biophotonics imaging technologyin the ewe and mare.

In Experiment 1, 30 pregnant ewes (~124 daysgestation) were assigned to one of 3 experimentalgroups. Following surgical scrub of the abdomen,ewes were inoculated trans-abdominally(ultrasound-guided either intra-cotyledon or intra-amniotic fluid) with 1 ml sterile broth alone(Control), or with either a low (Low, 1.2–4.0 x 106

CFU) or high (High; 5.6–20 x 106 CFU) dose of E.coli transformed with the pAK1-lux plasmid (E.coli-lux). The plasmid (11,904 bp) used is a broad-host-range cloning vector with numerous plasmidreplicons. A maximum of 30% of bacteria ejectedthe lux plasmid within the first 24 h posttransfection, while 70% retained and maintainedthe plasmid over an 8-day test period. Followinginoculation, ewes were monitored continuously forsigns of pre-term delivery. Trans-abdominal

ultrasonography was performed every 24 h forconfirmation of fetal viability over a 7-day intervalpost inoculation. Rectal temperatures wererecorded twice daily and blood collected byvenipuncture daily for cortisol (C) andprogesterone (P4) analysis. Lambs from ewes thatpre-term delivered within the 7-day interval wereimaged immediately. If ewes did not pre-termdeliver within the 7-day interval, euthanasia wasperformed on the 7th day and lambs recovered.Following euthanasia of ewes, the uterus andlambs or pre-term lambs were subjected tobiophotonic imaging using a Berthold/NightOwlcamera for detection of lux-expressing bacteria(photon emission) over a 5 min period and singleframe accumulation to determine pathogen tissuelocalisation. Subsequent to intact uteri and wholeanimal imaging, lambs were dissected and specificorgans imaged including heart, lungs, liver, gastro-intestinal (GI) tract and brain. Uterine and fetalfluids and fluids from GI tract, stomach andbladder were imaged for presence of emittingbacteria. All fluid samples were analysed todetermine total bacteria counts (CFU/ml). Pre-termdelivery in ewes occurred between 48 and 120 hpost inoculation. Of the 10 control ewes, 2 (20%)pre-term delivered and 8 carried normalpregnancies to Day 7 post inoculation (sterilebroth) when lambs were recovered. While a mixedbacterial growth was found in fetal fluid from oneof the pre-term fetuses, no photon-emittingbacteria were detected in any tissue or fluids fromcontrol lambs. Of the Low and High infected ewes,6 (60%) and 7 (70%) of the ewes pre-termdelivered, respectively. Imaging revealed in mostlambs that pre-term delivered photon-emittingbacteria were observed in the lungs, stomach, GI

14


tract, bladder, and in uterine fluids, but not in theheart or liver. No photon-emitting bacteria weredetected in brain tissue from any of the lambsdelivered or recovered from E. coli-lux-infectedewes. Cultures of fetal stomach, bladder anduterine fluids confirmed presence of photon-emitting bacteria.

In Experiment 2, 2 pony mares (late gestation)were inoculated trans-amniotically with E. coli-lux(1 x 106 CFU) to determine pathogen progressionand localisation in fetal foal tissues using the sameimaging approach as in Experiment 1. Fetuseswere collected 24 and 40 h post inoculation forimaging. Lux-expressing bacteria were found at 40h post inoculation in the lungs, GI tract, nares andsinuses but not in the brain, heart or liver. Thesedata clearly demonstrate the power of

bioluminescence imaging technology in detectingthe progression and localisation of pathogensinvolved in placentitis and pre-term delivery indomestic species.

In conclusion, these studies demonstrate thatbiophotonics and real time imaging provide anovel but valuable means of understanding thepathogenesis of bacteria associated with placentitisand pre-term birth in living mammalian species.Development of the pregnant ewe or mare as amodel for monitoring pathogen progression duringplacentitis in women has real potential.

ACKNOWLEDGEMENTS

Supported by USDA-NRI Grant #2003-35201-13841 and MSU-SRI Grant #341080.

15


ALLANTOIC CATHETERISATION OF THEPREGNANT MARE: DEVELOPMENT OF THETECHNIQUE AND PRELIMINARY FINDINGS

S. K. Lyle, D. Paccamonti, J. D. Hubert, J. R. Johnson, R. C. Causey* andB. E. EiltsDepartment Veterinary Clinical Sciences, Louisiana State University, School of Veterinary Medicine,Baton Rouge, Louisiana, 70803; *Animal and Veterinary Sciences, University of Maine, Orono, Maine,USA

Inflammation of the equine chorioallantois isproposed to cause increases in pro-inflammatorycytokines. Increased mRNA expression of IL-1,IL-6, and IL-8 in the chorioallantois and higherconcentrations of PGF2α and PGE2 in allantoicfluid were observed in mares transcervicallyinoculated with Streptococcus equi subsp.zooepidemicus compared to non-inoculated mares;however, no differences were detected in theconcentrations of soluble TNF-α, IL-1 and IL-6between groups. Placental infection causeselevations of pro-inflammatory cytokines in fetalfluids in other animal models (rhesus monkey,rabbit and mouse) and women. This unexpecteddiscrepancy emphasises the need for a non-invasive method of allantoic catheterisation forreliable sampling to study equine feto-placentalphysiology and pathology. The short-term goal ofthe present study was to identify a catheter thatcould be laparoscopically placed into pregnantmares and would maintain patency for a minimumof 7 days. The long-term goal is to use this cathetersystem to investigate the pathophysiology of fetaldemise for a variety of gestational diseases. Mareswere not fasted and received only sedation andregional local anesthesia pre-operatively.

During the first year of the study, 4 catheterdesigns were tested. Four mares were catheterisedsuccessfully with a commercially available humannephrostomy catheter, although biochemicalconfirmation that the fluid composition wasallantoic was only performed in 3 mares. Catheterswere patent for 5.6 days, with a range of 5–6 days;however, contamination of the allantoic catheterusually occurred by Day 5.

During the second year of the study severalrefinements were made to improve patientcomfort and reduce the risk of contamination,and 12 mares were successfully catheterisedduring 30 laparoscopies. These objectives wereoptimised by incorporating a subcutaneousaccess port system with an indwelling Huberneedle, and clipping and aseptically preparing theflank 24 h before surgery and again immediatelyprior to surgery. Fluid samples were obtained atsurgery, 4, 8, 12, 16, 20, 24, 28, 36, 42, 50 h postoperatively, and then daily until fetal delivery.Biochemical and cytological analysis of eachsample verified the nature of the fluid asallantoic, amniotic, peritoneal, or an admixtureof allantoic and amniotic fluid. For all mares,catheters were indwelling for a mean of 9.6 daysand were patent for 8.3 days. Of a potential 188samples, in only 16 instances could no fluidsample be obtained. The last 4 maresinstrumented were induced to deliver at Day 8following surgery; samples were obtained fromall from all of these mares at each of the specifiedtime points. Fluid from 2 of these mares waspurely of allantoic origin, while the other 2 maresshowed evidence of admixture of the allantoicand amniotic compartments. The latestrefinement of this procedure was the applicationof laparoscopic ultrasound to identify theallantoic compartment, thereby avoidingamniotic puncture. Ultrasound-guided,laparoscopic catheterisation of the allantoicspace will provide a reliable means ofinvestigating the pathophysiology of variousdiseases of equine pregnancy.

16


TROPHOBLAST CELL PHAGOCYTOSIS: AN IN VIVOSTUDY OF EARLY EVENTS IN ESTABLISHMENT OFEQUINE BACTERIAL PLACENTITIS

T. Infernuso, S. Fubini, J. Cheetham, H. Erb, D. Volkmann, R. Gleed and D. H. Schlafer

College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA

INTRODUCTION

Bacterial placentitis is a common cause of equineabortion. Before bacteria can establish placentalinfection, they first must breach a layer oftrophoblast cells that line the outer surface of thechorioallantoic membrane. Careful microscopicexamination of placental tissues from equineabortuses reveals that bacteria are occasionallyfound within the cytoplasm of trophoblast cells inareas where allantoic invasion and secondaryinflammation has not yet occurred, suggesting thattrophoblast cells had phagocytised bacteria towhich they are exposed. These observations led tothe working hypothesis that one of the earliestevents associated with establishment of bacterialplacentitis is phagocytosis of bacteria (or othermicrobes) present on the endometrial surface.Once bacteria have gained entry into placentaltissues, infection then quickly spreads, incitingtissue damage and inflammation that lead to fetalcompromise and abortion.

MATERIALS AND METHODS

The hypothesis that trophoblast cells phagocytisematerial present on the surface of thechorioallantois was tested by placement offluorescent microspheres in small chamberscreated surgically by separation of theendometrium and placenta. Three mares in theirlast trimester of gestation were used. Mares wereanaesthetised and a suspension containing 3 x 109

1 µm fluorescent carboxylate-modifiedmicrospheres (Molecular Probes, Eugene, Or) wasinjected through a small catheter temporarilyplaced in a small chamber, approximately 2 cm2,

that had been surgically created as describedabove. Uterine and placental tissues werecollected when mares were euthanised 4 h, 4 days,and 36 days later; samples were either fixed informalin or snap frozen. Frozen sections wereexamined by ultraviolet microscopy. Fixed tissueswere paraffin embedded, stained with H+E andexamined by light microscopy.

RESULTS

Microspheres were easily detected in the spacebetween the endometrium and chorioallantois insamples collected at 4 h and 4 days. In the 4 daysamples, large numbers of spheres were present inmacrophages around suture material and along theincision line in the uterine wall. Large numbers ofmicrospheres were found within the cytoplasm oftrophoblast cells but only rarely were spheresfound within endometrial epithelial cells lining thechambers. Trophoblast cells had much greaterphagocytic activity than did endometrial epithelialcells.

CONCLUSIONS

Trophoblast cells lining the equine placentareadily phagocytise particulate material. Thisexperimental evidence suggests that trophoblastcells play a role in the earliest stages oftransplacental infections. This experimentalapproach holds promise for more detailed,sequential, quantitative studies of mechanismsassociated with movement of particulate materialinto trophoblast cells and transmission ofmicrobial agents from mothers, across theirplacentas to their fetuses.

17


IDENTIFICATION OF NOVEL BIOMARKERS OFINTRA-AMNIOTIC INFECTION BY PROTEOMICPROFILING

M. G. Gravett

Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland, Oregon and Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton,Oregon, USA

Despite improvements in obstetrical care, pre-term birth remains the major obstetrical problemin developed countries. Sub-clinical or occultintra-amniotic infection (IAI) is a major cause ofpre-term birth, responsible for more than 50% ofextreme pre-term birth, where the highestproportion of neonatal deaths and seriouscomplications occur. Unfortunately, most womenin pre-term labour associated with occult intra-amniotic infection are refractory to standardtocolytic therapy. Moreover, antibiotic therapy hasnot prevented pre-term delivery in most studies,possibly because the patient subgroup with earlyIAI that might benefit from therapy is identifiedtoo late or not at all. Recent advances inproteomics present a new opportunity to examinethe global, or differential, expression of proteins intissues or fluids. The proteins or peptides that aredifferentially expressed in a disease orpathological state are well suited for thedevelopment of convenient, rapid, sensitive, andspecific diagnostic assays. In this study proteomicprofiling methods were used to discover novelbiomarkers for sub-clinical IAI in an experimentalnon-human primate model of IAI and in a cohortof pregnant women in pre-term labour.

Surface-enhanced laser desorption-ionisation/time-of-flight mass spectrometry (SELDI-TOF),gel electrophoresis, and tandem massspectrometry (MS/MS) were used to characteriseamniotic fluid peptides in 19 chronicallyinstrumented pregnant rhesus monkeys before andafter experimental IAI. Candidate biomarkerswere identified by liquid chromatography MS/MSand confirmed by Western blot. Amniotic fluidpeptide profiles identified in experimental IAIwere subsequently tested in a cohort of 33 womenin pre-term labour and delivery associated with

sub-clinical IAI (n=11), pre-term labour anddelivery without IAI (n=11), and pre-termcontractions with subsequent term delivery(n=11).

Protein expression profiles in amniotic fluidshowed unique signatures of overexpression ofpolypeptides in the 3- to 5-kDa and 10- to 12-kDamolecular weight ranges in all animals afterinfection and in no animal prior to infection. Inwomen, the 10- to 12-kDa signature was identifiedin all 11 patients with subclinical IAI, in 2 of 11with pre-term delivery without IAI, and in 0 of 11with pre-term labour and term delivery withoutinfection (P<0.001). Peptide fragment analysis ofthe diagnostic peak in amniotic fluid identifiedcalgranulin B and a unique fragment of insulingrowth factor binding protein 1, which were alsoexpressed in maternal serum. Mapping of otheramniotic fluid proteins differentially expressed inIAI identified several immunoregulators notpreviously described in amniotic fluid.

This proteomics-based characterisation of thedifferential expression of amniotic fluid proteinsin IAI identified a distinct proteomic profile in anexperimental primate chorioamnionitis model thatdetected subclinical IAI in a human cohort withpre-term labour. This diagnostic proteinexpression signature, complemented byimmunodetection of specific biomarkers inamniotic fluid and in maternal serum, hasapplication in the early detection of IAI. This mayallow for the timely diagnosis and treatment ofpre-term labour associated with subclinical IAI.

ACKNOWLEDGEMENTS

Supported by NIH AI42490, HD06159, RR00163,and 1U24DK5870.

18


MOLECULAR PATHOGENESIS OF THE GENITALMYCOPLASMAS

M. R. Peltier

Director of the Perinatal Biology Laboratory-Division of Maternal-Fetal Medicine, Department ofObstetrics, Gynecology and Reproductive Sciences, UMDNJ-Robert Wood Johnson Medical School,New Brunswick, New Jersey, 08901, USA

Pre-term premature rupture of membranes(PPROM) and pre-term labour (PTL) are theleading causes of perinatal morbidity andmortality. Although the aetiology of PPROM andPTL are often uncertain, they have beenfrequently linked to sub-clinical or clinicalinfection in the reproductive tract. During normalpregnancy, the production of pro-inflammatorycytokines such as IL-1β, IL-6, and TNF-α issuppressed. In the presence of infection, however,microorganisms increase the production of pro-inflammatory cytokines at the maternal-fetalinterface. These cytokines, in turn may promotePPROM and PTL by increasing apoptosis inplacental cells, increasing the production ofmatrix metalloproteinases or increasingprostaglandin secretion.

Much of the work with regard to how intra-uterine infection may cause PTL has been limitedto models that make use of lipopolysaccharide(LPS). Although valuable, they represent onlyinfections caused by Gram-negative bacteria suchas Escherichia coli. The majority of intra-uterineinfections, however, are caused by the genitalmycoplasmas Ureaplasma urealyticum andMycoplasma hominis. These small organisms,unlike E. coli, lack cell walls and hence LPS aswell. Therefore, models that use LPS poorlyrepresent infections caused by genitalmycoplasmas that must have different virulencefactors.

Therefore, we have focused our research onpurifying and characterising the pro-inflammatory components of these organisms. Wehave found that Triton X-114 detergent extractsfrom M. hominis and U. urealytcium are potentstimulators of TNF-α production by humanmacrophages. Extracts from M. hominis activatedToll-like receptor (TLR)-2 but not TLR-4, thepattern recognition receptor for the lipid Acomponent of LPS. The pro-inflammatoryactivity of M. hominis was stable to heating,partially reduced by proteinase K digestion, andcompletely abrogated by alkaline hydrolysis.Enrichment of the activity with SDS-PAGE andreverse-phase chromatography led to the isolationof a 29 kDa protein. Detergent extracts from U.urealyticum, however, were able to interact withboth TLR-2 and TLR-4. The pro-inflammatoryactivity of U. urealyticum was partially sensitiveto heating but completely removed by alkalinehydrolysis or proteinase K digestion and theactivity was associated with proteins at 40 and 66kDa. These studies suggest that the activity ofgenital mycoplasmas contain a factor(s) that maybe similar to the Macrophage-activatinglipoprotein-2 (MALP-2) or its precursor (MALP-404) that was previously isolated fromMycoplasma fermentans. Further work is neededto purify the activity to homogeneity and to testits ability to cause pre-term birth in an animalmodel.

19


THE USE OF BETAMETHASONE TO ADVANCEFETAL MATURATION IN THE EQUINE

D. Christiansen*,† G. Olsen†, J. Smith*, R. Hopper*, M. M. LeBlanc**

and P. Ryan*,†

*Department of Pathobiology and Population Medicine, College of Veterinary Medicine;†Department of Animal and Dairy Sciences, Mississippi State University; **Rood and Riddle Equine Hospital, Lexington, Kentucky, USA

An important adjunct to the treatment ofplacentitis is the attempt to induce precocious fetalmaturation to ensure fetal viability in the event ofa potential pre-term delivery. In women,betamethasone sodium phosphate (Celestone) ordexamethasone is commonly used to advance fetalmaturation in high-risk pregnancies. Variousattempts to induce precocious maturation of theequine fetus have met with mixed results.Intrafetal injections of adrenocorticotropichormone (ACTH), betamethasone (BMS), anddexamethasone (DEX) have been shown toadvance equine fetal maturation, but areassociated with a high (~30%) incidence ofspontaneous abortion. Currently, although DEX isthought to be effective only when given very latein gestation, practitioners utilise high doses ofDEX (100 mg) given to the dam 4 daysconsecutively in an attempt to initiate equine fetalmaturation. Therefore, the aim of this pilot studywas to ascertain whether fetal maturation of thefoal could be induced precociously by maternalinjection with BMS. Thirteen Quarter Horsemares received either saline (SAL, n=5), or BMSinjections (im) at 12 mg (n=3), 24 mg (n=3) or 30mg (n=2) on Day 305, 306 and 307 of gestation.Delivery was induced (20 IU oxytocin injections)on Day 320 in 5 mares (2 x 12 mg, 2 x 24 mg and1 x 30 mg). Other BMS-treated mares were notinduced due to a poor response to treatment. Foalblood samples were collected at 0, 24 and 48 h andanalysed for cortisol (C), progesterone (P4),thyroxine (T4) and triiodothyronine (T3)concentrations. Blood cell counts at 0 h and 24 hpost partum were also used as markers of foalmaturity. All SAL and non-induced treated maresfoaled at term without complications. Only onemare in the 24 mg group delivered normallyfollowing induction. This mare had a mild

nocardioform placentitis found onhistopathological examination that may havecaused precocious maturation of that fetus. Allother induced mares required assistance.Complications associated with the prematureinduction of parturition in mares included onemare failing to exhibit maternal behaviour, onehaving a cervical tear and one having a uterine andcervical tear. Two foals (24 mg) survived toweaning while the remaining 3 (2 x 12 mg and 1 x30 mg) were euthanised within 48 h due todysmaturity and failure to respond to clinicalsupport. Consequently, data was analysed as SAL(non-induced, n=5), treated non-induced (T-NI,n=3) and treated induced (T-I, n=5). Serum C wasundetectable in 4 of 5 T-I foals with P4 valuesgreater (12.5. ± 3.1 ng/ml, P>0.05) at 0 hcompared to SAL and T-NI foals (9.1 ± 1.9, 8.7 ±1.7 ng/ml, respectively). Serum T4 and T3 valueswere higher in SAL and T-NI compared with T-Ifoals (27.0, 25.9 and 20.8 µg/dl for T4,respectively; and 6.0, 7.3 and 4.0 ng/ml for T3,respectively). White blood cells at 0 h were greaterin T-I than T-NI (P<0.05) and SAL (P<0.1) foalswith a neutrophil:lymphocyte ratio of 4:7, 7:2 and4:1, respectively. SAL and T-NI treated maresfoaled at term without complications, with BMSadvancing delivery by 7–14 days in T-NI mares.

In summary, while BMS did not acceleratefetal maturation adequately to successfully inducepre-term delivery of foals, maternal BMStreatment appeared to advance delivery date innon-induced mares. As an advanced delivery datewould likely indicate an effect on fetal maturation,a second study was undertaken to determine if thisobservation was significant.

Sixteen Quarter Horse type mares were pairedby gestation length. Mares were injected on Days305, 306, and 307 of gestation with either saline or

20


24 mg BMS and allowed to foal. Serum wascollected from mares on Day 305 and then 3times weekly until foaling for P4 analysis.Whole blood and serum was collected from thefoals at birth for a CBC and P4 analysis andserum was collected at 24 h of age for additionalP4 analysis and evaluation of passive transfer.All foals were clinically mature at birth;however, although not statistically significant,there was a trend toward decreasing gestationlength in the treated mares versus the control

mares (339 ± 3.8 vs. 345 ± 12.2 daysrespectively). Further studies need to beconducted to determine if increased dose orprolonged treatment may enhance this effect aswell as to ascertain any differences which mayoccur in the presence of placentitis.

ACKNOWLEDGEMENTS

Supported by Mississippi Agriculture andForestry Experiment Station.

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SESSION 3:

Pathological findings

Chairman: C. Sanchez

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23


EQUINE PLACENTAL PATHOLOGY: THE COMMONAND THE NOT SO COMMON

N. M. Williams, J. M. Donahue, D. C. Bolin, R. C. Giles, L. R. Harrison,C. B. Hong, C. B. Jackson, K. B. Poonacha and M. L. Vickers

University of Kentucky, 1490 Bull Lea, Box 14125, Lexington, Kentucky 40512, USA

INTRODUCTION

The University of Kentucky Livestock DiseaseDiagnostic Centre (LDDC) is located in the heartof the central Kentucky bluegrass region with itsnumerous horse breeding operations. Each yearthe centre receives several hundred fetuses,placentas, and stillborn foals for necropsyexamination, making it one of the most valuablesites for prospective/retrospective studies ofnaturally occurring equine feto-placental diseaseand pathology.


A computer-based search of the LDDC caserecords from the past 3 foaling seasons: 2003,2004, and 2005 was conducted. All equine caseswith a standard diagnosis of placentitis wereidentified. The case reports were reviewed and thenumber of cases of placentitis, the time ofoccurrence of each case, the pattern of the grosspathology, the bacteria isolated and the sites wherethe bacteria were isolated were investigated. Thecases were grouped by total yearly cases, time ofoccurrence, pathology, and bacterial isolates.

RESULTS

The most common diagnosis in fetuses andplacentas submitted to the LDDC is placentitis.The majority of the cases result from bacterialinfection of the placental membranes, often withconcurrent fetal infection. Over the 3 foalingseasons, 619 cases of placentitis were identified.There were 372 cases in foaling year 2003, 127cases in 2004, and 120 cases in 2005. Few cases ofplacentitis were diagnosed between the months of

June through September. The number of casesbegan to increase in October and reached a peakduring the early months of the year, followed by atapering toward the end of the foaling season (Fig1). This pattern of gradual increase and declinewas consistent for the seasons that were studied.

The case review revealed that, of the 619cases, 302 had gross lesions on the placentasuggestive of placentitis. Based on the narrativedescription, 52 cases were determined to representascending placentitis. The typical gross lesionsconsisted of areas of discoloration and thickeningof the chorion with variable surface exudatepresent. Of the 619 cases, 205 were diagnosed ashaving placentitis by finding inflammation withinthe placental tissues on microscopic examinationwith no suggestive gross changes. These casestypically had low numbers of neutrophils andmononuclear cells in the chorionic villi and sub-villous stroma with degeneration of the chorionicepithelium. Cases with gross changes suggestiveof placentitis usually had more severeinflammation and degeneration of the chorioniccells, often with congestion and haemorrhage.

Bacteriological cultures were typicallyperformed on the placenta (chorion) and fetallung, liver, and stomach fluid. Of the 619placentitis cases, significant bacteria were isolatedin 364 cases (59%). Bacteria were also culturedfrom sites in addition to the placenta in 139 cases(38% of the cases in which bacteria were isolated).Cultures in 118 cases were overgrown bysaprophytic microorganisms and 136 cases hadbacteria considered to be nonpathogenic. Therewere 28 cases of mycotic placentitis diagnosed.These were typically ascending placentitis casesand the fungus was usually classified asAspergillus sp.

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Fig 1: Placentitis cases by month.

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Fig 2: Nocardioform placentitis by foaling year.

The most commonly isolated bacteria were thegram-positive branching filamentous bacilli.These partially classified bacteria are placed in thebroad group nocardioform actinomycetes. Theseare the bacteria associated with nocardioformplacentitis, a common diagnosis in centralKentucky but only rarely reported elsewhere.

The next most common group of bacteria wasthe streptococci, followed by a variety of otherorganisms, including the recently reported (Bolinet al. 2004). Cellulosimicrobium cellulans (Table1). Rare and uncommon isolates included single

cases of Aeromonas hydrophila, Dermatophiluscongolensis, Salmonella paratyphi, andRhodococcus equi infection.

Nocardioform placentitis is an importantcause of fetal loss in central Kentucky. It ischaracterised by inflammation of the chorion inthe area of the base of the placental horns oranterior body of the placenta, with accumulationof thick mucoid material on the surface (Donahueand Williams 2000). This form of placentitis wasfirst recognised in Kentucky in 1986. The yearlycaseload since 1991 typically averages from 20 to

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40 cases (Fig 2). However, several years have hadfew cases and other years have produced manymore cases.

Nocardioform placentitis does not involve thecervical star area. It appears to begin as a singlefocus of infection that expands and enlarges overtime. The central area is characterised by low-level chronic inflammation (mononuclear) anddestruction of villi, while the periphery has moreabundant active inflammation and cellulardegeneration. Bacteria are scarce in the centre ofthe lesion but are typically numerous in theperiphery. The bacteria do not appear to beinvasive and are not cultured from the fetal fluidsor tissues (other than the chorion). Nocardioformplacentitis appears to injure the fetus by causingplacental insufficiency. The time of entry and theroute of infection are not known.

Nocardioform placentitis usually results in nooutward manifestation of infection and placentitisin the mare; however, some mares exhibitpremature mammary gland development andlactation. There are 4 possible outcomes tonocardioform placentitis: the mare may abort, themare may carry the fetus to term but produce astillborn foal, the foal may be born alive(premature or full term) but weak andcompromised, or the mare may deliver a normalfoal. Following delivery the mare usually clearsthe infection rapidly, rebreeds normally, and doesnot appear to be at increased risk for subsequentabortion. Occurrence on a particular farm issporadic.

Several strains of the nocardioformactinomycetes causing nocardioform placentitishave been classified. These include Crossiellaequi, Amycolatopsis kentuckyensis, Amycolatopsislexingtonensis, and Amycolatopsis pretoriensis(Donahue and Williams 2002; Labeda et al. 2003).

Cases of placentitis indistinguishable grosslyfrom nocardioform placentitis, but associated withother types of bacteria, have been identified. Overthe 3 foaling seasons, 32 such cases wereidentified. Cultures and histopathology werenegative for nocardioform-type bacteria and, in allcases tested, PCR tests for C. equi andAmycolatopsis were negative. Bacteria isolatedfrom these cases included Pantoea agglomerans,Cellulosimicrobium cellulans, Pseudomonas spp.,Enterobacter spp., Enterococcus spp. andStaphylococcus spp. In addition to the placenta,these bacteria were also sometimes cultured fromthe fetal tissues.

Unusual cases of chronic amnionitis resultingin thickening and contraction of the amnion werediagnosed occasionally. There were 21 cases in2003, 4 in 2004, and one in 2005. Some cases alsohad allantochorion and cord lesions; however,most involved only the amnion. The cause isusually not known but bacteria were isolated inabout 25% of the cases, suggesting possiblechronic bacterial infection. Some cases wereassociated with premature meconium passage.

Mare Reproductive Loss Syndrome (MRLS)resulted in numerous early gestation and late termabortions in 2001 and 2002. Funisitis, rarely seen

TABLE 1: Bacterial isolates from placentitis cases

Number of isolates Organism

111 (26%) Gram-positive branching filamentous bacteria81 (19%) Streptococcus (zooepidemicus, equisimilis, spp.)68 (16%) Leptospira spp. 45 (11%) E. coli27 (6%) Pantoea (Enterobacter) agglomerans20 (5%) Cellulosimicrobium (Oerskovia) cellulans15 (4%) Pseudomonas spp.12 (3%) Enterobacter cloacae11 (3%) Enterococcus spp.7 (2%) Stenotrophomonas (Pseudomonas) maltophilia7 (2%) Staphylococcus spp.5 (1%) Actinobacillus equuli4 (1%) Citrobacter freundii2 Corynebacterium spp.1 Aeromonas hydrophilia1 Dermatophilus congolensis1 Salmonella paratyphi1 Rhodococcus equi

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with other causes of equine abortion andplacentitis, was a characteristic feature of abortedlate term fetuses. The affected cords wereoedematous with dull gray discoloration androughening of the surface. Only the amnioticsegment was affected, changes were not noted inthe allantoic portion of the cord. Microscopically,stromal oedema, haemorrhages, inflammatory cellinfiltrates, and colonisation of the surface bybacteria were observed. Placentitis (chorionitis)was diagnosed in some cases but was much lesscommon than funisitis (Williams et al. 2003).Placentitis was not typically suspected on grossexamination and, microscopically, theinflammation was mild. Chorionic inflammationor bacterial colonisation was not observed. Theinflammation was predominately neutrophilic andlocated in the stroma, often within the extraembryonic coelom.

DISCUSSION

Other studies have found that most infections ofthe equine feto-placental unit are bacterial inorigin with Streptococcus zooepidemicus beingthe most commonly isolated pathogen (Giles et al.1993; Hong et al. 1993). In this report the group ofnocardioform actinomycetes was most commonlyisolated. The streptococcal bacteria were thesecond largest group. Isolates of Streptococcuszooepidemicus and Streptococcus equisimilis wereapproximately equal in number, however. Inagreement with a previous report (Hong et al.1993) that found that bacteria were isolated in68% of placentitis cases, the present study foundbacteria in 59% of cases.

This study found that approximately 60% ofplacentitis cases had gross lesions on the placentasuggesting placentitis. The finding thatapproximately 40% of the placentitis cases werediagnosed microscopically without gross lesionssuggests that many cases are the result of acute,rapidly progressing infections.

Placentitis cases that on gross examinationappeared to be nocardioform placentitis but wereactually the result of infection by other bacteriawere occasionally seen. This indicates that the

development of this particular form of placentitisis not uniquely a property of nocardioformbacteria, but bacteria that also cause more classicalplacentitis can, under certain unknown conditions,produce placentitis morphologically identical tonocardioform placentitis. Therefore, naming thisform of placentitis based on the bacteria isprobably inaccurate and a morphologic namewithout specification of causative agent may bemore appropriate, ie, focal mucoid placentitis.

REFERENCES

Bolin, D.C., Donahue, J.M., Vickers, M.L., Giles, R.C.,Harrison, L., Jackson, C., Poonacha, K.B., Roberts,J.E., Sebastian, M.M., Sells, S.E., Tramontin, R. andWilliams, N.M. (2004) Equine abortion andpremature birth associated with Cellulosimicrobiumcellulans infection. J. Vet. Diagn. Invest. 16, 333-336.

Donahue, J.M. and Williams N.M. (2000) Emergingcauses of placentitis and abortion. Vet. Clin. NorthAm. Equine Pract. 16, 443-456.

Donahue, J.M., Williams, N.M., Sells, S.F. and Labeda,D.P. (2002) Crossiella equi sp. nov., isolated fromequine placentas. Int. J. Sys. Evol. Microbiol. 52,2169-2173.

Giles, R.C., Donahue, J.M., Hong, C.B., Tuttle, P.A.,Petrites-Murphy, M.B., Poonacha, K.B., Roberts,A.W., Tramontin, R.R., Smith, B. and Swerczek,T.W. (1993) Causes of abortion, stillbirth, andperinatal death in horses: 3,527 cases (1986-1991).J. Am. Vet. Med. Assoc. 203, 1170-1175.

Hong, C.B., Donahue, J.M., Giles, R.C. Jr, Petrites-Murphy, M.B., Poonacha, K.B., Roberts, A.W.,Smith, B.J., Tramontin, R.R., Tuttle, P.A. andSwerczek, T.W. (1993) Etiology and pathology ofequine placentitis. J. Vet. Diagn. Invest. 5, 56-63.

Labeda, D.P., Donahue, J.M., Williams, N.M., Sells, S.F.and Henton, M.M. (2003) Amycolatopsiskentuckyensis sp. nov., Amycolatopsislexingtonensis sp. nov., and Amycolatopsispretoriensis sp. nov., isolated from equine placentas.Int. J. Sys. Evol. Microbiol. 53, 1601-1605.

Williams, N.M., Bolin, D.C., Donahue, J.M., Giles,R.C., Harrison, L.R., Hong, C.B., Poonacha, K.B.,Roberts, J.F., Sebastian, M.M., Smith, B.J., Smith,R.A., Swerczek, T.W., Tramontin, R.R. and Vickers,M.L. (2003) Mare Reproductive Loss Syndrome:Gross and histopathological correlates. Proc. of theFirst Workshop on Mare Reproductive LossSyndrome. Eds: D.G. Powell, A. Troppman, and T.Tobin. Lexington, Kentucky. Sess. 2, pp. 24-25.

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GROSS AND MICROSCOPIC PATHOLOGY OFPLACENTAS AND FOAL ORGANS INEXPERIMENTALLY INDUCED EQUINE ASCENDINGBACTERIAL PLACENTITIS

M. B. Calderwood Mays*, M. M. LeBlanc** and M. L. Macpherson†

Florida Veterinary Pathology, Gainesville, Florida; **Rood and Riddle Equine Hospital, Lexington,Kentucky; †College of Veterinary Medicine, University of Florida, Gainesville, Florida, USA

From 1999–2005, placentas were examined from36 pony mares with experimentally inducedbacterial placentitis and one pony mare withbacterial placentitis contracted after experimentalsurgical intervention. Tissues from 32 foalswhich were born dead or euthanised due to poorhealth shortly after birth were examined at thesame time. (5 foals lived). The experimentallyinfected mares had been inoculated with 107 or108 colony forming units of Streptococcus equisubspecies zooepidemicus between 270 and 295days of gestation. Sixteen of the mares had beentreated with various antibiotics and anti-inflammatory drugs for varied periods of timeafter inoculation.

Pathology samples from the placentasincluded cervical star region, body, gravid horn,and nongravid horn of the chorioallantois, amnion,and umbilical cord. Foal tissues included lung,liver, kidney, spleen, and adrenal. The mostcommon lesion was necrotising placentitis in thecervical star region of the chorioallantois withspread to the uterine body region, seen in 34 cases.Bacteria were recognised in the necrotic tissue in

30 cases and in one additional case in whichnecrotising placentitis developed only at a catheterentrance site. Funiculitis of the umbilical cord wasrecognised in 19 cases. Bacteria were found on orin the peripheral layers of the umbilical cord in 14cases. Localised amnionitis was recognised in 10cases. In 20/32 cases bacteria were found inairways and alveoli of the foals’ lungs. The lungtissue exhibited early pneumonia in 7 cases.

Cultures collected at the time of parturitioncorrelated well with the bacteria seen in the tissueshistologically – Streptococcus equi subspecieszooepidemicus in 11 cases, with a gram negativerod (Escherichia coli, Klebsiella) as a dualinfection with Streptococcus in 3 cases, a gramnegative rod alone in 5 cases, and Nocardia in onecase. Bacterial septicemia was never recognised ina foal. All organs except lung were histologicallyuninflamed and uninfected. The authors suspectthe organisms reached the lung by traversing thechorioallantois (in the cervical star region of theplacenta in all but one case) and travelling to theamniotic fluid along the umbilical cord, to beaspirated by the foal.

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29


SESSION 4:

Treatment strategiesfor infection in lategestation

Chairman: M. M. LeBlanc

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31


INNATE IMMUNITY AND MICROBIOLOGICALLYINFORMED APPROACHES TO PREVENTION ANDTREATMENT OF INTRAUTERINE INFECTIONDURING PREGNANCY

J. A. McGregor, O. Equils, M. Wilson and S. Witkin

School of Medicine at USC Los Angeles, California; Geffen School of Medicine at UCLA, Los Angeles,California; Weil School of Medicine, Cornell University New York, USA

AIMS

1) This study aimed to analyse systems biology(molecular-clinical) approaches to prevent and/ortreat intra-uterine infection/inflammation duringpregnancy; 2) Highlight clinical and researchopportunities to optimise reproduction outcomesimpaired by infection/inflammation.


Selected current research and ‘best’ clinical carepractices were reviewed regardingpericonceptional, embryonic, and perinatalimmune, microbiological, and inflammationinteractions affecting pregnancy andneonatal/childhood outcomes (pre-term birth, lowbirth weight, pre-term premature rupture ofmembranes and perinatal organ injury).

RESULTS

Selected ‘key’ pathophysiological areaspresented included: 1) mechanisms of intra-

uterine infection/inflammation; 2) evidence ofintra-uterine microbes and adverse fetal/maternaleffects (pre-term labour and birth, rupture ofmembranes, impaired growth and developmentand organ damage; 3) inter-relationships between maternal, fetal, and paternal bilogicrelationships; 4) evidence of improvedreproductive outcomes with appropriatedetection and treatment of abnormal hostmicrobial interactions, especially during earlygestation; 5) exploration of ‘best’ antimicrobialand other treatments to optimise prevention andtreatment strategies for preventing infection-inflammation caused prematurity and perinatalorgan system damage; 6) use of biomarkers to detect and monitor intra-uterineinfection/inflammation.

CONCLUSIONS

Comparative and systems-biology informedapproaches were presented to guide and informresearch and clinical care in order to optimisereproduction outcomes.

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NOVEL TREATMENT STRATEGIES FOR INFECTION-INDUCED PRE-TERM BIRTH: A NON-HUMANPRIMATE MODEL

M. G. Gravett

Department of Obstetrics & Gynecology, Oregon Health & Science University, Portland, Oregon andDivision of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, Oregon,USA

Pre-term birth and its consequences account for80% of perinatal deaths not attributed tocongenital malformations. Intra-amnioticinfections (IAI) are an important and potentiallypreventable cause of extremely premature births at24–30 weeks of gestation, where perinatalmortality and morbidity are high. Antibioticsalone, however, are not effective in preventinginfection-induced pre-term birth. Pro-inflammatory cytokines and prostaglandinsparticipate in the pathogenesis of infection-associated pre-term births. The mechanisms,however, by which infection and thecytokine/prostaglandin cascade lead toprematurity remain speculative and treatmentstrategies largely untested. It has beendemonstrated previously in non-human primates,in the absence of infection, that intra-amnioticinfusion of the pro-inflammatory cytokine IL-1βresults in increases in uterine contractility and pre-term delivery and that this contractility can beblocked by administration of cytokine immuno-suppressants (dexamethasone) or prostaglandinsynthesis inhibitors (indomethacin). Chronicallyinstrumented pregnant rhesus macaques weretherefore used to investigate the hypothesis thatdown-regulation of the inflammatory cascadetogether with antibiotic treatment would prolonggestation in the setting of infection.

Fifteen chronically instrumented pregnantrhesus monkeys with timed gestations receivedexperimental IAI by intra-amniotic inoculation of106 Group B streptococci (GBS) at 134–141 daysof gestation (term is 167 days). Amniotic fluid(AF) cytokines (IL-1β, IL-6, IL-8, TNF-α),prostaglandins (PGE2 and PGF2α), matrixmetalloproteinases (MMP-2, MMP-9), and uterinecontractility (hourly contraction area, or HCA, inmmHg s/h) were serially measured before and

after infection and/or intervention. Followinginfection and increases in uterine contractility, 4animals received no treatment, 4 animals receivedmaternal ampicillin (IV 30 mg/kg every 6 h), and5 animals received ampicillin plus maternal oralindomethacin (50 mg bid) plus maternaldexamethasone (IV 1 mg/kg every 6 h).

Following intra-amniotic infection with GroupB streptococci without treatment, there weresequential increases in AF pro-inflammatorycytokines (IL-1β, IL-6, IL-8, and TNF),prostaglandins (PGE2 and PGF2α), matrixmetalloproteinases (MMP-9), and uterine activity.Uterine activity increases occurred from pre-inoculation HCA levels of 0–100 mmHg s/h topeak levels of 10,000 to 20,000 mmHg s/h afterinoculation and led to delivery at an average of 37+ 9 h after infection.

Animals treated with antibiotics plusimmunosuppressants had significant prolongationof pregnancy following infection (213 + 50 h),when compared to those treated with antibioticsalone (81 + 28 h), or those receiving no treatment(37 + 9 h; P<0.01, ANOVA).

These data support the central role of the pro-inflammatory response in infection-induced pre-term birth and suggest that novelimmunomodulators in combination withantibiotics may be more effective than antibiotictherapy alone in the prevention of infection-induced pre-term birth. The chronicallyinstrumented pregnant rhesus monkey modelprovides insights into the pathophysiology ofinfection-induced pre-term labour, and willfacilitate development of rationale and effectivemedical strategies to prevent prematurity.

ACKNOWLEDGEMENTS

Supported by NIH AI42490.

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DETECTION OF GENTAMICIN AND PENICILLINCONCENTRATIONS IN ALLANTOIC FLUID OFPREGNANT PONY MARES BY IN VIVOMICRODIALYSIS

T. A. Murchie, M. L. Macpherson, M. M. LeBlanc, S. Luznar and T. W. Vickroy

College of Veterinary Medicine, Western University of Health Sciences, 309 E. Second Street,Pomona, California 91766-1854, USA

OBJECTIVES

Current treatment protocols for equine placentitisinconsistently prevent premature parturition. Littleis known about the transfer of drugs across theequine placenta, and treatment strategies arelargely empirical. The primary objective of thisstudy was to develop an in vivo microdialysistechnique for monitoring drug concentrations inallantoic fluid of pregnant pony mares followingsystemic drug administration.


The study was conducted on 5 pony mares in lategestation.

Microdialysis probes were inserted into theallantoic fluid using transabdominal ultrasoundguided allantocentesis. Penicillin G, gentamicin andflunixin were given iv and dialysate samples werecollected continuously over a 24 h period. In aseparate study, samples were collected from 2 ponymares following intracervical infection with aninoculum of Streptococcus equi subspecieszooepidemicus. Drug concentrations in dialysatefractions and serum samples were determined usinghigh performance liquid chromatography (penicillinG, flunixin) or an enzyme-linked immunosorbentassay (gentamicin). Descriptive data analysis wasperformed, all results reported as means ± standarderror, and minimum inhibitory concentrations(MIC) indicated for the 2 infected mares.

RESULTS

Penicillin G and gentamicin were detectedreadily in allantoic fluid of all 5 non-infected

mares, with peak concentrations of 9.8 ± 2.2mg/ml and 8.5 ± 3.1 mg/ml, respectively. Whencompared to drug concentrations in serum,penicillin G remained detectable in allantoicfluid for approximately twice as long.Gentamicin exhibited similar elimination profilesfrom serum and allantoic fluid. In addition,penicillin G achieved similar concentrations inthe allantoic fluid of the 2 infected mares (11.2mg/ml) as non-infected mares whereasgentamicin was present at much lowerconcentrations in infected mares (3.9 mg/ml).Furthermore, penicillin G concentrations in theallantoic fluid of the 2 infected mares exceededthe MIC appropriate for Streptococcus equisubspecies zooepidemicus (β-haemolyticstreptococci; MIC ≤ 0.12 mg/ml) tested in thisstudy. Gentamicin allantoic concentrations didnot reach the reported MIC values for certainGram negative organisms (Klebsiella spp., MIC≤ 10 mg/ml).

Flunixin was undetectable throughout theentire sampling period in allantoic fluid of allsubjects.

CONCLUSIONS

Microdialysis is a useful tool for short-termcontinuous monitoring of drug concentrations inequine allantoic fluid. The results demonstrate thatpenicillin G and gentamicin undergo effectiveplacental transfer in non-infected mares althoughthe pharmacokinetic profiles of individual drugsare notably different between serum and allantoicfluid. Therapeutic concentrations of penicillin Gwere observed in allantoic fluid of the 2 inoculatedmares.

34


PLACENTAL PENETRATION OF TRIMETHOPRIMSULFAMETHOXAZOLE AND PENTOXIFYLLINE INMARES WITH PLACENTITIS

S. Rebello, M. L. Macpherson, T. Murchie†, M. M. LeBlanc* andT. W. Vickroy††

Department of Large Animal Clinical Sciences and ††Department of Physiological Sciences, Collegeof Veterinary Medicine, University of Florida, Gainesville, Florida; †Western University of HealthSciences, Pomona, California; *Rood and Riddle Equine Hospital, Lexington, Kentucky, USA

Placentitis is the most prevalent cause of equineabortion, and remains a large source of economicloss to the breeding industry. Current treatmentmodalities have been inconsistent in preventinginfection-associated premature labour. Theprimary objective of this study was to determinethe ability of selected drugs to pass through theblood-placental barrier in normal mares and mareswith experimentally-induced placentitis. Theauthors hypothesised that placentitis would notalter the pharmacokinetic profiles or the freeconcentrations of selected therapeutic agents inallantoic fluid.

Ten late-gestational mares (276–300 days ofgestation) were used for this study. Placentitis wasinduced in 5 mares using an intracervicalinoculation of Streptococcus equi subspecieszooepidemicus (107 CFU) prior to samplecollection. Five mares served as uninfectedcontrols. Allantoic fluid drug concentrations weredetermined for all mares by in vivo microdialysis.Prior to drug treatments, a microdialysis probewas implanted in the allantoic cavity usingtransabdominal ultrasound guidance.Trimethoprim-sulfamethoxazole (TMP-SMZ, 30mg/kg, q 12 h, PO) and pentoxifylline (PTX, 8.5mg/kg, q 12 h, PO) were administered untilabortion or parturition for a maximum period of 2weeks. Microdialysate samples were collectedcontinuously for a period of 36 h in conscious,

free-standing mares. Drug concentrations wereanalysed by reverse-phase high performanceliquid chromatography with ultravioletspectroscopy. Peak drug concentrations, time-to-peak intervals, and total drug concentrations(reported as area-under-the-curve) were recordedas means ± standard error. Control versus infectedgroups were statistically compared using a non-parametric Wilcoxson Rank-Sum Test (P<0.05).Mean TMP and SMZ levels were compared to theminimum inhibitory concentration (MIC) reportedfor in vitro control of S. equi zooepidemicus.

All drugs exhibited placental transfer and weredetectable by microdialysis sampling. Nosignificant differences (P>0.05) were detectedbetween control and infected mares with regard topeak levels of TMP, SMZ, or PTX in allantoicfluid. Based upon reported MIC values against S.equi zooepidemicus, the levels of TMP and SMZin allantoic fluid should be sufficient to elicit anantibiotic effect for up to 4 h following eachtreatment in control and infected mares. It isunclear whether allantoic levels of PTX aresufficient to elicit any therapeutic action in vivo.Together, these data demonstrate that passage ofthe selected drugs through the blood-placentalbarrier is unchanged in mares withexperimentally-induced placentitis, and revealsnon-linear relationships between drug levels inplasma and allantoic fluid.

35


EXPERIMENTALLY INDUCED PLACENTITIS IN LATEGESTATION MARES WITH STREPTOCOCCUS EQUIZOOEPIDEMICUS: PREVENTION OF PRE-TERM BIRTH

P. Ryan*, J. Crouch*, D. Sykes*, K. Moulton*, D. Christiansen*, R. Hopper,R. Read, W. Bennett† and M. M. LeBlanc**

Department of Pathobiology and Population Medicine and *Department of Animal and DairySciences, Mississippi State University, Mississippi State, Mississippi; †Department of Obstetrics andGynecology, University of Mississippi Medical School, Jackson, Mississippi; **Rood and Riddle EquineHospital, Lexington, Kentucky, USA

Placental infection due to opportunistic pathogens(ie Streptococcus equi subspecies zooepidemicus(S. equi)) is the single most common cause ofabortion, stillbirth and premature delivery inhorses. The consequence of placentitis and pre-term delivery results in severe financial loss to theequine industry. Moreover, increasing evidencedemonstrates that placentitis increases pro-inflammatory cytokine expression leading topremature delivery. The authors’ workinghypothesis is that inhibition or blocking of the pro-inflammatory cytokine responses with the use of acombinatorial drug therapeutic strategy thatincludes antibiotics and known anti-inflammatorycytokine agents will reduce the incidence of pre-term labour in pregnant mares with uterineinfections. Thus, the objective of this pilot studywas to induce ascending placentitis and evaluatetwo therapeutic strategies to prevent cytokine-induced pre-term birth in late gestation mares.

To this end, in Experiment 1, 6 ponies (~ 290days gestation) were infected intracervically with~2 x 106 colony forming units (CFU) of a clinicalstrain of S. equi and assigned (n=3/group) toreceive either trimethoprim sulfamethoxazole(TMS; 30 mg/kg BW, q12h) alone or incombination with Regumate (altrenogest;TMS+R; 2.0 mg/50 kg BW, q24h). Drugsensitivity tests confirmed that this strain of S.equi was sensitive to TMS.

In Experiment 2, 12 late term-pregnant mareswere assigned to one of 3 experimental groups. Eight mares were inoculatedtranscervically S. equi (~2 x 106 CFU) as inExperiment 1 and were assigned to receive eitherantibiotics alone (TMS: 30 mg/kg BW, q12h) or in

combination with dexamethazone (TMS+DEX)while 4 mares served as non-infected, non-medicated controls (CON). DEX wasadministered daily over a 6 day period withdecreasing doses every 2 days from 40, 35 to 25mg, respectively. Blood samples were collectedprior to infection and at 12, 24, 48, 72 h postinfection and 3x/week thereafter until delivery formacrophage cytokine mRNA, relaxin andprogesterone (P4) analysis. Fetal and placentalwell-being was evaluated daily by transrectalultrasonography. Treatment (TMS, TMS+R orTMS+DEX) commenced upon initial signs ofvaginal discharge and/or placental changes. TMSwas maintained through to delivery and for 7 dayspost partum. Blood was collected from foals at0 and 24 h post partum for complete blood count(CBC) (neutophil: lymphocyte ratio),immunoglobulin G (IgG) and P4 analysis asindices of maturity. Placentaes and fetuses weresubmitted for necropsy and histopathology.

In Experiment 1 all 6 mares showed signs ofvaginal discharge and/or placental changes within36 h of inoculation. Three aborted 11, 12 and 14days post inoculation, 2 from the TMS+R groupand one from the TMS group; the remaining 3mares carried to near-term delivering viable foals.Mean birthweight of TMS and TMS+R foals was19.55 ± 1.82 and 17.55 ± 1.71 kg, respectively.Placental thickening increased (P< 0.001) from0.77 ± 0.04 cm at pre-inoculation to 1.17 ± 0.06cm at 48 h post inoculation. Pathology confirmedascending necrosuppurative placentitis andbronchopneumonia in aborted fetuses. Culture ofstomach contents of aborted fetuses revealedheavy growth of S. equi.

36


In Experiment 2 the mean gestational stage attime of S. equi inoculation was 297 ± 2.6 forinfected vs 307 ± 3.4 days for control mares. Theaverage stage of gestation at time of delivery forCON, TMS and TMS+DEX mares was 340 ± 7.6,319 ± 6.4 and 293 ± 3.7 days, respectively. AllCON mares delivered normal viable foals while 2of the inoculated mares aborted dead fetuses, onefrom each treatment group. Of the remaininginfected mares, 6 produced live pre-term foals (3from TMS, 3 from TMS+DEX) 5 of which wereviable while one from the TMS+DEX group waseuthanised due to poor viability indices. Mean daysat which pre-term delivery occurred for CON, TMSand TMS+DEX mares were -0.25 ± 7.6, 20.8 ± 6.3

and 37.3 ± 3.2 days, respectively. Mean birthweightof CON, TMS, TMS+DEX foals was 47.2 ± 1.8,41.0 ± 3.6 and 34.9 ± 2.3 kg, respectively.Pathology of placentaes from infected maresshowed varying degrees of lesions and oedemaconsistent with ascending placentitis. In conclusion,due to low numbers, data is inconclusive as towhether the combinatorial therapy of TMS+R orTMS+DEX is more effective in the prevention ofpre-term delivery than TMS alone. Cytokine andblood hormone analyses are pending.

ACKNOWLEDGEMENTS

Supported by MSU-SRI Grant #341080.

37


PROGESTINS AND PRE-TERM BIRTH: WHAT ISTHE EVIDENCE AND DOES IT MAKE SENSE?

M. A. Elovitz

University of Pennsylvania, 1353 BRB II/III, 421 Curie Boulevard, Philadelphia 19104, USA

Over half a century ago, progestins were used toprevent pre-term birth. In 1990, a meta-analysissuggested that these agents may be of benefit butfurther research was warranted. In 2003, arandomised control trial demonstrated that aprogestational agent, 17-α hydroxyprogesterone(17-P), significantly decreased the rate of pre-term birth in high-risk patients. However, sinceprogesterone levels in pregnancy are alreadyquite high, it remains unclear by whatmechanisms progestational agents can prevent

pre-term birth in human pregnancy. Animalstudies have begun to elucidate the mechanismsby which progestins may decrease pre-term birth.These studies suggest that progestatinal agentsmay prevent pre-term birth through non-traditional progesterone-receptor mediatedevents. A better understanding these mechanismswill allow for more appropriate use of thesedrugs in human pre-term birth and thus,ultimately help to decrease pre-term birthwithout incurring undue fetal or maternal harm.

38


THE ROLE OF ENDOGENOUS AND EXOGENOUSPROGESTAGENS IN PREGNANT MARES

J. C. Ousey, P. D. Rossdale*, E. Houghton†, L. Grainger†, L. Palmer*

and A. L. Fowden

University of Cambridge, Department of Veterinary Medicine and Department of Physiology,Development and Neuroscience, Cambridge; *Rossdale & Partners, Beaufort Cottage Stables, HighStreet, Newmarket, Suffolk; †Horseracing Forensic Laboratories, Newmarket Road, CambridgeshireCB7 5WW, UK

Progesterone (P4) and related compounds(progestagens) are necessary for maintenance ofpregnancy in the mare. Before about 100 daysgestation, progestagens are produced by theovaries, removal of which causes a decline incirculating concentrations and abortion. After thistime, progestagens are produced increasingly bythe feto-placental tissues, and concentrations onlydecline, in healthy mares, at parturition (term 320and 360 days). P4 is one of about 10 progestagenswhich have been quantified in maternal and fetalplasma using gas chromatography-massspectrometry (Holtan et al. 1991). Theprogestagen metabolic pathway and enzymesinvolved are shown in Figure 1. Becauseprogestagens are synthesised within the uterus,umbilical concentrations of most progestagens aresubstantially higher than in the maternalcirculation. The fetus produces large quantities(3.6 increasing to 9.9 mmol/min in late gestation)of pregnenolone (P5) probably from the adrenals,although whether the adrenal glands possesssufficient P450scc enzyme activity to produce allthis P5 remains unknown. The P5 is metabolisedby the utero-placental (UP) tissues into P4, 5α-dihydroprogesterone (5α-DHP) and 20α-hydroxy-5-pregnan-3-one (20α-5P). Small quantities (<0.5mmol/min) of P4 are excreted exclusively into theumbilical circulation so consequently P4 is rarelyfound in the maternal circulation. In contrast,excretion of 5α-DHP and 20α-5P by the UPtissues, is divided between the umbilical anduterine circulation. From mid-gestation, 5α-DHPexcretion into the uterine circulation predominatesbut, during the last month of pregnancy, asignificantly greater proportion of 5α-DHP isexcreted into the umbilical than uterine blood(Ousey et al. 2003). Whether this redirection of5α-DHP has any relevance to parturition remains

unclear. Any 5α-DHP returned to the fetus ismetabolised into other progestagens which areexcreted back to the UP tissues. There is no nettransfer of progestagens from the mare across theUP tissues into the fetus.

Because little P4 is present in maternalplasma, measurement of progesterone by ELISAor RIA tends to reflect the total progestagenconcentrations and clearly values vary accordingto the specificity of the progesterone antibody andtype of assay system used. Concentrations alsovary between Thoroughbreds and Ponies althoughthe individual progestagens detected in bothbreeds are similar (Rossdale et al. 1991).Therefore, it is important to establish normalvalues for healthy pregnant mares for a givenassay system. Both fetal and maternal plasma totalprogestagen concentrations increase graduallyover gestation with a large rise occurring duringthe last few weeks pre-partum, associated withincreased production of P5 by the fetus. In the last24–48 h prior to delivery, fetal (and maternal)progestagen concentrations decline concurrentwith a rise in fetal plasma cortisol. This indicatesa switch from progestagen to glucocorticoidproduction due to activation of the enzyme, 17α-hydroxylase, in the fetal adrenal.

Damage to the placenta whether throughplacentitis or other types of placental disease,stimulates a precocious rise in total progestagenconcentrations in maternal plasma (Fig 2). Asimilar increase usually can be induced in healthymares by experimental manipulation of theplacenta causing placental damage and/orinfection (Rossdale et al. 1991; Stawicki et al.2002). This precocious rise in total progestagensprobably occurs through activation of the fetalHPA axis in response to the chronic placentalinsult, because total progestagens in maternal

39


Cholesterol

P5

P4

5α-DHP

3β5P

ββdiol

P5ββ

20α−OHP420β−OHP4

20α−5P

βαdiol

Putative pathway

Abreviations

P5: PregnenoloneP5ββ: 5-pregnene-3β,20β-diolP4: Progesterone20αOH-P4: 20α-hydroxyprogesterone20βOH-P4: 20β-hydroxyprogesterone5αDHP: 5α-pregnane-3,20-dione3β-5P: 3β-hydroxy-5α-pregnan-20-one20α5P: 20α-hydroxy-5-pregnan-3-oneβα-diol: 5α-pregnane-3β,20α-diolββ-diol: 5α-pregnane-3β,20β-diol

Enzymes

1. P450scc,sidechain cleavage (F&P)2. 3β-HSD, 3β-hydroxysteroid dehydrogenase (F&P)3. 5α-reductase (E&F)4. 3β-oxidoreductase (P)5. 3-oxidase (P)6. 20β-reductase (F)7. 20α-reductase (E)

P = Placenta

E = Endometrium

F = Fetus

Fig 1: Progestagen synthesis in the pregnant mare and fetus.

25

20

15

10

5

0240 250 260 270 280 290 300 310 320 330 340 350 360

Gestational age (days)

Pla

sma

tota

lpro

gest

agen

s(n

g/m

l)

Fig 2: Maternal plasma progestagen concentrations in healthy, pregnant Thoroughbred mares (n=19,mean ± sem) and in 4 pregnant Thoroughbred mares with clinical problems.

Healthy (n=19)

Colic

Uterine rupture

Placentitis 1

Placentitis 2

5

1

6

5

3

2

4

7

6

7

7

6

4

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plasma can rise in response to intra-fetal injectionwith adrenocorticotrophic hormone (ACTH). Incontrast, when pregnant mares have more acuteclinical problems, not involving the placentadirectly, eg colic or uterine rupture, maternalplasma progestagen concentrations usually declineassociated with rapid fetal demise and beforeactivation of the fetal HPA axis (Fig 2). GC-MSanalysis of the progestagens present in maternalplasma from clinical cases with placentitis (n=7)has revealed elevated concentrations of almost allof the progestagens including P4 and/or P5 overthe weeks prior to abortion or premature delivery(Ousey et al. 2005). These results indicateincreased production of P5 by the fetus andincreased metabolism of P5/P4 into theirmetabolites by the UP tissues, despite placentalinfection and possible fetal compromise. Incontrast, in 2 clinical cases of mares aborting withextensive villous poverty and placental oedema,respectively, total progestagens were raisedassociated with increased plasma P4 butconcentrations of most progestagen metabolites,including 5α-DHP, were either normal or evenreduced. In these cases it appears that damage tothe UP tissues was such that metabolism of P4 intoother progestagens was compromised, leading totransfer of P4 into the maternal circulation.

Exogenous progestagen therapy is oftenprescribed for mares at risk from abortion. Formares with placentitis this therapy appearsunnecessary because their progestagenconcentrations are already elevated. However,administration of synthetic P4 (altrenogest)combined with antibiotic and anti-inflammatorytreatments, has extended gestational length andimproved foal viability (Troedsson and Zent2004), although at present there appears to be noscientific rationale for this treatment. Indeed otherprogestagens, for example 5α-DHP, may be morebiologically important in maintaining quiescenceof the equine myometrium. 5α-DHP binds morestrongly than P4 to the uterine P4 receptor and ispresent in higher concentrations than P4 in theumbilical circulation (Chavatte-Palmer et al.2000). However, studies, in vitro, usingmyometrial tissue from pregnant mares hasdemonstrated that neither P4 nor 5α-DHP canprevent or alter the frequency of myometrialcontractions induced with oxytocin (Ousey et al.2000). But how these in vitro analyses relate to thesituation in vivo, particularly in mares with clinical

problems, remains unclear. Few studies haveinvestigated the effects of exogenous progesteroneor its synthetic counterpart, altrenogest, onendogenous progestagens in late gestation mares.Administration of P4 to healthy mares at 318 daysgestation significantly shortened gestationallength compared with control mares, but no sucheffect was observed in mares receiving eitherprogesterone or altrenogest for 10 days from 300days gestation (Alm et al. 1975; unpublishedobservations); there was an increase in P4following progesterone treatment only, but allother progestagens remained unchanged followingeither treatment, compared with controls.Differences between these 2 studies may relate tobasal progestagen concentrations at the time oftreatment. Administration of altrenogest to 2mares with placentitis did not appear to alterprogestagen profiles, (or outcome) compared with5 mares with placentitis that received noaltrenogest (Ousey et al. 2005).

It has been suggested that P4 may altermyometrial contractility by regulating UPsynthesis of prostaglandins (PG) in the mare, asoccurs in other species. Local production of P4from P5, via 3β-hydroxysteroid dehydrogenase(3β-HSD) in the fetal trophoblast, may act in aparacrine fashion to regulate production ofprostaglandin dehydrogenase (PGDH) located inthe adjacent maternal microcotyledons (Han et al.1995). This may be one pathway wherebyexogenous P4 could control PG production andmyometrial contractility. However, progestagensat similar concentrations to those found at the endof pregnancy inhibit placental 3β-HSD in vitroand blockade of 3β-HSD activity in vivo and invitro, causes a transient decline in P4 and otherprogestagens although parturition was notinduced and 5α-DHP concentrations recoveredwithin 1.5 h (Fowden and Silver 1987; Chavatteet al. 1997). Therefore, these results indicate thathigh concentrations of progestagensparadoxically could lead to a decline incirculating levels and possibly to early delivery.Moreover, they do not appear to support the casefor progesterone therapy, particularly in maresthat may already have elevated endogenousprogestagen concentrations. Clearly, moreinformation is needed about effects ofprogestagen therapy on steroid metabolism withinthe feto-placental unit in healthy and sickpregnant mares.

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REFERENCES

Alm, C.C., Sullivan, J.J. and First, N.L. (1975) Theeffect of a corticosteroid (dexamethasone),progesterone, oestrogen and prostaglandin F2α ongestation length in normal and ovariectomisedmares. J. Reprod. Fert. 23, 637-640.

Chavatte, P, Holtan, D, Ousey, J.C. and Rossdale, P.D.(1997) Biosynthesis and possible role ofprogestagens during equine pregnancy and in thenewborn foal. Equine vet. J. 24, 89-95.

Chavatte-Palmer, P., Duchamp, G., Palmer, E., Ousey,J.C., Rossdale, P.D. and Lombes, M. (2000)Progesterone, oestrogen and glucocorticoidreceptors in the uterus and mammary gland of mid-to late gestation mares. J. Reprod. Fert. 56, 661-672.

Fowden, A.L. and Silver, M. (1987) Effects of inhibiting3β-hydroxysteroid dehydrogenase on plasmaprogesterone and other steroids in the pregnant marenear term. J. Reprod. Fert. 35, 539-545.

Han, X., Rossdale, P.D., Ousey, J.C., Holdstock, N.B.,Allen, W.R., Silver, M., Fowden, A., McGladdery,A.J., Labrie, F., Belanger, A., Ensor, C.M., Tai, H-H.and Challis, J.R.G. (1995) Localisation of 15-hydroxy prostaglandin dehydrogenase (PGDH) andsteroidogenic enzymes in the equine placenta.Equine vet. J. 27, 334-339.

Holtan, D.W., Houghton, E., Silver, M., Fowden, A.L.,Ousey, J.C. and Rossdale, P.D. (1991) Plasmaprogestagens in the mare, fetus and newborn. J.Reprod. Fert. 44, 517-528.

Ousey, J.C., Freestone, N., Fowden, A.L., Mason. W. T.

and Rossdale, P.D. (2000) The effects of oxytocinand progestagens on myometrial contractility invitro during equine pregnancy. J. Reprod. Fert. 56,681-689.

Ousey, J.C., Forhead, A.J., Rossdale, P.D., Grainger, L.,Houghton E. and Fowden, A.L. (2003) Theontogeny of uteroplacental progestagen productionin pregnant mares during the second half ofpregnancy. Reproduction, 69, 540-548.

Ousey, J.C., Houghton E., Grainger, L., Rossdale, P.D.and Fowden, A.L. (2005) Progestagen profilesduring the last trimester of gestation inThoroughbred mares with normal or compromisedpregnancies. Theriogenology, 63, 1844-1856.

Rossdale, P.D., Ousey, J.C., Cottrill, C.M., Chavatte, P.,Allen, W.R. and McGladdery, A.J. (1991) Effects ofplacental pathology on maternal plasmaprogestagens and mammary secretion calciumconcentrations and on neonatal adrenocorticalfunction in the horse. J. Reprod. Fert. 44, 579-590.

Stawicki, R.L., Ruebel, H., Hansen, P.J., Sheerin, B.R.,O’Donnell, L.J., Lester, G.D., Paccamonti, D.L.,LeBlanc, M.M. (2002) Endocrinological findings inan experimental model of ascending placentitis inthe mare. Theriogenology, 58, 849-852.

Troedsson, M.H.T. and Zent, W.W. (2004) Clinicalultrasonographic evaluation of the equine placentaas a method to successfully identify and treat mareswith placentitis. In: Proc. Workshop on the EquinePlacenta, Eds: D.G. Powell, D. Furry, G. Hale,Kentucky Agricultural Experiment Station,Lexington, Kentucky, USA pp 66-67.

42


OMEGA-3 (N-3) FATTY ACID NUTRITION:OPTIMISING REPRODUCTIVE AND EARLY LIFENEUROLOGICAL PERINATAL OUTCOMES

J. A. McGregor and M. Harris

Keck School of Medicine at USC Los Angeles, California; Colorado State University. Fort Collins,Colorado, USA

AIMS

This study aimed to analyse aspects of n-3polyunsaturated fatty acid (PAFAs) nutrition andpregnancy performance and selected offspringneurological outcomes.


Information was analysed regardingDocosahexanoic acid (DHA), eicosapentaenoicacid (EPA) and related substances obtained in foodor diet supplementation which affects reproductiveperformance and/or growth, development, andneurological functioning in offspring.

RESULTS

Systems-based research demonstrates multiple,protean biologic effects n-3 polyunsaturated fattyacids (PUFA) nutrition and metabolism as well asinteracting substances including n-6 PUFAs, transfats, and n-6 FAs. ‘Nutrigenomic’ studies show

altered epigenetic and patterning functioning ofmultiple genes (ras, others) affecting form, andfunction of maternal and fetal organ systems.‘Proteinomic’ and physiological analysesdemonstrate multiple effects of n-3 nutrition,including cell membrane fluidity, receptor activity(insulin, IGF axis), and CNS functions. Pregnancyevaluation in both animals and humans showalteration of gestational length (DHA, EPA).Developmental investigations show improvedfunctioning of CNS and special sense (vision)performance parameters. Increasing evidencedemonstrates n-3 eficacy in treatment and possibleprevention of post partum and other types ofdepression.

CONCLUSIONS

Rapidly accumulating information justifies furtherbasic and clinical research into improvingpregnancy and offspring and outcomes by meansof enhanced fatty acid nutrition during pregnancyand lactation.

43


SESSION 5:

Effects of the neonate

Chairman: D. Paccamonti

44


45


PLACENTITIS ASSOCIATED NEONATAL PROBLEMSAND THE EFFECT OF THERAPEUTICINTERVENTIONS

J. E. Palmer, P. L. Sertich, B. I. Cornman, P. A. Wilkins and R. C. Boston

Department of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University ofPennsylvania, Kennett Square, Pennsylvania, USA

Disruption of the intra-uterine environment maybe the initiator of many serious neonatal diseases.The inflammatory response may directly affectplacental sufficiency or the inflammatory cascadeaccompanying the placentitis may have secondaryadverse consequences for the fetus. The authorsreport on a preliminary retrospective study of therelationship between placentitis and neonataldiseases and the effect of therapy.

Clinician notes from foals and their maresfrom 2000 through 2005 were reviewed. Caseswith complete fetal membrane evaluation by thesecond author (PLS) and foal evaluation by thefirst author (JEP) were included. Data wasanalysed through logistic regression. A p-value of5% was used to separate chance from factor drivendifferences in outcomes, and a p-value interval of5% and 10% was used to highlight the presence oftrends.

One hundred and eight (108) cases wereidentified. When all cases were considered, therewas no association of Neonatal Encephalitis (NE),Neonatal Nephropathy (NN) or NeonatalGastroenteropathy (NG) with placentitis or pre-partum treatment regardless of the occurrence ofplacentitis. However, when treatment ofplacentitis was considered, NE, NN and NG weresignificantly more likely to occur in foals born tomares with untreated placentitis. There was a trendfor bacteremia to be associated with placentitisand it was also more likely in foals with untreatedplacentitis. Treatment of mares with any

combination of antimicrobials, NSAIDs orprogestins significantly protected foals against thedevelopment of NE, NN and NG. No significantbenefit of treatment was seen in preventing sepsisor bacteremia. Mares with placentitis were morelikely to have a normal foal if treated withantibiotics, NSAIDs or progestins.

There is a strong association of placentitis andneonatal diseases (NE, NN and NG) but only inuntreated cases. This strong association supportsthe hypothesis that placentitis is the cause of thesediseases. In addition pre-partum treatment of themare for placentitis appears to strongly protectagainst development of these diseases. Commonlyutilised therapy seemed to contribute to thisprotective affect. Surprisingly, treatment,independent of the presence of placentitis, showeda trend to protect against sepsis suggesting thatsomething other than placentitis which respondedto treatment could pre-dispose the foal to sepsis.Alternately, treatment of the mare might decreasethe exposure of the neonatal foal to factors thatpredispose to sepsis. These trends will be exploredfurther as more cases are added to this data set.Although bacteremia was more likely in foals frommares with untreated placentitis, pre-partumtreatment of the mares did not protect frombacteremia. Treatment of mares with placentitissignificantly increased the odds of having a foalwithout any of the neonatal problems. Mares withsuspect placentitis should be treated pre-partum toprevent development of common neonatal diseases.

46


EVALUATION OF COAGULATION IN NEONATALFOALS BORN TO MARES WITH AND WITHOUTPLACENTITIS

A. Bentz, P. L. Sertich, R. C. Boston, P. A. Wilkins and J. E. Palmer

Department of Clinical Studies, New Bolton Center, University of Pennsylvania, Kennett Square,Pennsylvania, USA

The association of sepsis, inflammation andcoagulation derangement is well documented inveterinary and human patients (Short 2004). In theinitial stages of sepsis, inflammatory cytokinesactivate tissue factor (TF) while TF inhibitors maybe decreased. This leads to unregulatedcoagulation and complications such asdisseminated intravascular coagulation (DIC)which may be an important contributor toMultiorgan Dysfunction Syndrome (MODS).

Although perinatal coagulopathy has beenassociated with congenital infections, there is scantinformation in the literature regarding coagulationin critically ill neonatal foals or neonatal foals bornto mares with placentitis (Barton et al. 1995;Barton et al. 1998; Bentz et al. 2002). This studyexplores the association of abnormal coagulationparameters and the occurrence of placentitisaccessed through fetal membrane examination.

Prospective evaluation of coagulationparameters in 68 neonatal foals admitted to ourNICU has been previously reported. A subgroup of14 foals born to mares with gross and histologicalplacental examinations is reported here. Eightmares were treated with anti-inflammatory,antimicrobial and/or progestin therapy. Five ofthese mares had no significant findings on placentalexamination and 3 were classified as placentitis.

Parturition was attended and blood collectedby direct venipuncture at birth, 24 h and 48 h afterdelivery for platelet count, prothrombin time (PT),partial thromboplastin time (PTT), fibrindegradation products (FDPs), fibrinogen andantithrombin. DIC was defined as 3/6 parametersoutside normal range during one sampling period.Placentas were collected immediately afterparturition and evaluated by gross and histologicalexamination.

Foals were categorised into 4 groups: septicshock, sepsis, other diseases or healthy (Bone etal. 1992; Abraham et al. 2000). Outcome wasdefined as survival to discharge or death prior todischarge. Coagulation parameters, clinicaldiagnosis and outcome were analysed inrelationship to presence or absence of placentitis.

Fetal membranes of 6/14 foals were classifiedas abnormal with 4/14 being retained, 1/14 havinggross placentitis only, 1/14 having histologicalplacentitis only (as well as being retained) and1/14 with both gross and histological placentitis.Foals born to these mares were classifiedclinically as follows: 2/6 suffering septic shock,2/6 having sepsis, 1/6 with other problems and 1/6healthy. Foals born with normal fetal membraneswere classified as follows: none having septicshock, 2/8 having sepsis, 3/8 having otherproblems and 3/8 being healthy. There was onenon-survivor from the non-placentitis group. Nofoal exhibited clinical evidence of bleeding;however, 3 foals born to mares with normalplacental examination were classified in DIC.13/14 foals survived to discharge.

Analysis of coagulation parameters inrelationship to placental examination and maternaltherapeutic intervention was performed usinglogistic regression. Differences between groupsand/or sampling intervals were evaluated using theKruskal-Wallis test. Significant coagulationresults for the foals included decreased PT andplatelet count if mares were treated. Additionalinvestigations are needed.

ACKNOWLEDGEMENTS

This study was funded by The InternationalArabian Horse Association.

47


REFERENCES

Abraham, E., Matthay, M.A., Dinarello, C.A., Vincent,J.L., Cohen, J., Opal, S.M., Glauser, M., Parsons, P.,Fisher, C.J. and Repine, J.E. (2000) Consensusconference definitions for sepsis, septic shock, acutelung injury and acute respiratory distress syndrome:time for a reevaluation. Crit. Care Med. 28, 232-235.

Barton, M.H., Morris, D.D., Crowe, N., Collatos, C. andPrasse, K.W. (1995) Hemostatic indices in healthyfoals from birth to one month of age. J. Vet. Diagn.Invest. 7, 380-385.

Barton, M.H., Morris, D.D., Norton, N. and Prasse, K.W.(1998) Hemostatic and fibrinolytic indices in

neonatal foals with presumed septicemia. J. Vet.Intern. Med. 12, 26-35.

Bentz, A.I., Wilkins, P.A., MacGillivray, K.C., Barr, B.S.and Palmer, J.E. (2002) Thrombocytopenia in twothoroughbred foals with sepsis and neonatalencephalopathy. J. Vet. Intern. Med. 16, 494-497.

Bone, R.C., Balk, R.A., Cerra, F.B., Dellinger, R.P.,Fein, A.M., Knaus, W.A., Schein, R.M. and Sibbald,W.J. (1992) Definitions for sepsis and organ failureand guidelines for use of innovative therapies insepsis: The ACCP/SCCM Consensus ConferenceCommittee. Chest 101, 1644-1655.

Short, M.A. (2004) Linking the sepsis triad ofinflammation, coagulation and suppressedfibrinolysis to infants. Adv. Neonatal Care 4, 258-273.

48


EQUINE NEONATAL SEPSIS: SHORT AND LONGTERM OUTCOMES

L. C. Sanchez and G. D. Lester*

Department of Large Animal Clinical Sciences, University of Florida College of Veterinary Medicine,Gainesville, Florida, USA; *School of Veterinary and Biomedical Sciences, Murdoch University, Perth,Western Australia

Equine neonatal sepsis has been implicated as themajor cause of morbidity and mortality in theyoung foal. Both prenatal and post natalconditions have been implicated as predisposingfactors for the development of sepsis. Prenatalfactors, such as placentitis, dystocia, andpremature placental separation, were reported in24% of bacteremic foals in a recent study (Stewartet al. 2002).

The purpose of the current study was toevaluate short- and long-term survival rates, andidentify changes in microbial isolates and theirantimicrobial susceptibility patterns over the last22 years. All available medical records fromequine neonates (30 days or less at first admission)admitted to the Hofmann Equine NeonatalIntensive Care Unit at the University of Floridabetween 1982 and 2004 were used and data wereentered into a computerised database format.From these records, information regarding foalsdiagnosed with sepsis was obtained, includingclinical data, clinicopathological data, and short-term outcome. Race records for Thoroughbredfoals and their 3 closest maternal siblings werecollected. Information regarding the microbialisolates, including source, timing relative toadmission, and antimicrobial sensitivity patternswas also recorded. For purposes of this study,septic foals were considered those from which apositive blood culture was obtained. Foals weregrouped by decade (1982–1989; 1990–1999;2000–2004) for some analyses. This study was

expanded from data reported previously (Sanchezand Lester 2000) to include foals from 2000–2004.

Positive blood cultures were recorded from393 foals during the study period. Overall, 467microbial isolates were obtained from foals atadmission, and 41 isolates were obtained afteradmission. Throughout the study period, E. coliwas the predominant organism isolated. Over 50%of all isolates were categorised as enteric gram-negative rods, although this percentage decreasedslightly each decade. Gram positive organismsaccounted for approximately 20–25% of allisolates throughout the study. Interestingly, thepercentage of gram-positives increased in the2000-2004 group when considering isolatesobtained after admission only. Overall short-termsurvival was 57%, with the survival rateincreasing steadily by decade. Long-termoutcome, as measured by the percentage of startersand winners, of short-term survivors comparedfavourably with that of their maternal siblings.

REFERENCES

Sanchez, L.C. and Lester, G.D. (2000) Equine neonatalsepsis: Microbial isolates, antimicrobial resistance,and short and long term outcomes. Proc. ACVIMForum 18, 223-224.

Stewart, A.J., Hinchcliff, K.W., Saville, W.J.A., Jose-Cunilleras, E., Hardy, J., Kohn, C.W., Reed, S.M.and Kowalski, J.J. (2002) Actinobacillus spbacteremia in foals: Clinical signs and prognosis. J.vet. Int. Med. 16, 464-471.

49


THE OUTCOME OF FOALS BORN TO MARESTREATED FOR PLACENTITIS

B. S. Barr

Rood and Riddle Equine Hospital, PO Box 12070, Lexington, Kentucky 40580-5070, USA

Equine placentitis is the most common cause ofabortion and stillbirth in the mare (Giles et al.1993). Pre-term birth or the birth of a weak andcompromised foal are also the sequalae ofplacentitis. Any of these scenarios result ineconomic losses to the equine owner. Pre-termbirth can result in complex complications in theequine species because fetal maturation occurswithin the last few days of gestation. Thus, thefoal is often born alive, but may require extensivehospitalisation or maybe small in size and lesslikely to become an athlete. Clinical signs ofequine placentitis include premature lactation andvaginal discharge. If these clinical signs areobserved, diagnostics are performed to assess thelower and upper reproductive tract and theviability of the fetus. Diagnostics includetransrectal sonogram, transabdominal sonogramand vaginal speculum examination. The mare ismost frequently treated with antimicrobials, non-steroidal anti-inflammatories and tocolytics(Macpherson 2005). If the foal is born pre-term orterm, but weak and compromised the prognosis isassumed to be poor. The purpose of the studyreported here was to determine the short-term andlong-term outcome of foals born to mares treatedfor placentitis. Medical records at Rood andRiddle Equine Hospital from 1986–2005 wereevaluated to identify those cases in whichplacentitis was the diagnosis based on pathologyreport of the placenta and that information wasavailable on the mare and foal. Information thatwas obtained from the mare’s record includedhistory or reason for evaluation (prematurelactation, vaginal discharge), diagnosticsperformed (transabdominal ultrasound, transrectalultrasound and vaginal speculum examination)and treatment (type and length). Informationobtained from the foal’s record included viability

of the foal at birth, gestational length, physicalcharacteristics (body size), health of the foal,length of treatment if the foal requiredhospitalisation and diagnosis.

Thirty mares/foals were identified that met theinclusion criteria. Twenty-eight of the 30 caseswere Thoroughbreds, one was a Standardbred andthe other was an American Saddlebred. The mostcommon reason for admission and evaluation ofthe mare included premature lactation or vaginaldischarge (22/30 cases). Five of the 30 mares wereadmitted with a history of placentitis, definitiveclinical signs were unable to be obtained from therecords. Two mares were admitted with acompromised neonate that was less than 24 h old.One mare was admitted with a history of abnormalstage one labour. A total of 15 mares had atransabdominal ultrasound performed, 10 of the15 were noted to be abnormal. Twenty-seven ofthe mares had been treated for placentitis prior toparturition, whereas 3 had not. Medication usedfor treatment included: antimicrobials(trimethoprim/sulfa, penicillin, gentamicin,metronidazole), anti-fungals (diflucan), anti-inflammatories (flunixin meglamine,phenylbutazole), and tocolytics (regumate,isoxsuprine, clenbuteral). The trimethoprim-sulfaand regumate combination was the mostcommonly administered treatment. Only 13 of the30 placentas submitted for pathological evaluationresulted in growth of a microorganism. The mostcommon isolated organism was Norcadioform(8/13) (gram-positive bacillus) with otherorganisms cultured including Aspergillus, Pantoea(Enterobacter) agglomernas, Streptococcuszooepidemicus and E. coli.

Twenty-nine of the 30 foals were born alive,whereas one was dead. Of the 29 born alive, 23lived to discharge and 6 foals died or were

50


euthanised. The physical characteristics werebased on the foal having a small or average bodysize as noted by the clinician in the record.Average size was considered to be approximately110 pounds for a Thoroughbred foal. Twenty-threeof the 30 foals were noted to be small in size andseven were noted as being average size. Sixteen ofthe 29 foals were born healthy (not requiring anytreatment or extensive hospitalisation). Thehealthy foals ranged from 311–347 days ofgestation. There were 13/29 unhealthy/compromised foals that required treatment andhospitalisation. The gestational age of theunhealthy foals ranged from 299–355 days ofgestation. Clinical diagnosis of the unhealthy/compromised foals included: prematurity (4),septicemia (2), neonatal encephalopathy (1),pneumonia (1), failure of passive transfer (1),congenital abnormalities (1), neonatalencephalopathy/septicemia (1), prematurity/septicemia (1) and prematurity/failure of passivetransfer (1). Four of these foals died and 2 wereeuthanised, the rest were alive at discharge. Thelength of hospitalisation for the foal ranged from2–14 days with a cost estimated cost of $2,000 to>$10,000. Twenty foals were of racing age. Racerecords were obtained through the BloodstockResearch Information Services or US TrottingAssociation. Fifteen of the 20 foals had one start,with the range of starts 1–26. The earnings rangedfrom $0–$35,949 with the average $13,367.

Nocardioform placentitis is found commonlyin Central Kentucky, but rarely reportedelsewhere. This is caused by gram-positivefilamentous branching bacteria (Donahue andWilliams 2000). Eight mares were diagnosed withnocardioform placentitis and all foals were viableat birth. Seven of the 8 were alive at discharge andone was euthanised due to economic reasons. Therange of gestation for these foals was 311–355days. Seven of the 8 foals were born with a smallbody size, whereas one was of average size. Three

of the 7 were born healthy, but 3 were prematureand one was septic. The 4 compromised foalsrequired extensive treatment and hospitalisation.Each of these 8 mares had been treated forplacentitis prior to parturition. All but one wastreated with the following combination:trimethoprim/sulfa, regumate and flunixinmeglamine. The other mare was treated withpenicillin, gentamicin and regumate. Seven ofthese foals were of racing age. Four of the 7started but only one won a race.

Overall this retrospective study identified thata majority of the foals were viable at birth butsmall in size. If the foal was born healthy, butsmall in size, the foal was discharged from thehospital. If the foal was born unhealthy and smallin size only 45% were discharged. Out of thepopulation of small foals that are of racing age, 9out of 14 raced in at least one race, whereas 6 ofthe 7 average size foals started. Looking at thegroup as a whole, these foals did not earn much asa racehorse, which confirms our assumptions thatthese foals do not excel as a racehorse. Some ofthese foals may have gone on to be riding horsesand may have excelled in that capacity, but thisinformation is not known. This study reveals somepreliminary information about foals born to marestreated for placentitis, but further data needs to beobtained to comfortably give a prognosis on theoutcome of these foals.

REFERENCES

Donahue, J.M. and Williams, N.M. (2000) Emergentcauses of placentitis and abortion. Vet. Clin. NorthAm. Equine. Pract. 16, 443-456.

Giles, R.C., Donahue, J.M., Hong, C.B., Tuttle, P.A.,Petrites-Murphy, M.B., Poonacha, K.B., Roberts,A.W., Tramontin, R.R., Smith, B. and Swerczek,T.W. (1993) Causes of abortion, stillbirth, andperinatal death in horses: 3,527 cases (1986–1991).J. Am. Vet. Med. Assoc. 203, 1170-1175.

Macpherson, M.L. (2005) Treatment strategies for mareswith placentitis. Theriogenology 64, 528-534.

51


APPENDIX

52


53


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55


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


LIST OF PARTICIPANTS

BONNIE BARR

United States

AMY BENTZ

United States

MARON CALDERWOOD MAYS

United States

DAVID CHRISTIANSEN

United States

MICHAL ELOVITZ

United States

MICHAEL GRAVETT

United States

MICHELLE LEBLANC

United States

SARAH LYLE

United States

MARGO MACPHERSON

United States

JOSH MAGSIG

United States

JAMIE MCGREGOR

United States

TRACY MURCHIE

United States

JENNY OUSEY

United Kingdom

DALE PACCAMONTI

United States

JON PALMER

United States

MORGAN PELTIER

United States

GENE PRANZO

United States

PETER RYAN

United States

CHRIS SANCHEZ

United States

DONALD SCHLAFER

United States

NEIL WILLIAMS

United States

72


AUTHOR INDEX

BARR, B.S., 49

BENNETT, W. see RYAN, P. et al.

BENTZ, A. et al., 46

BOLIN, D.C. see WILLIAMS, N.M.et al.

BOSTON, R.C. see BENTZ, A. et al.;PALMER, J.E. et al.

CALDERWOOD MAYS, M.B. et al.,27

CAUSEY, R.C. see LYLE, S.K. et al.

CHEETHAM, J. see INFERNUSO, T.et al.

CHRISTIANSEN, D. et al., 19 and seeRYAN, P. et al.

CORNMAN, B.I. see PALMER, J.E.et al.

CROUCH, J. see RYAN, P. et al.

DONAHUE, J.M. see WILLIAMS,N.M. et al.

EILTS, B.E. see LYLE, S.K. et al.

ELOVITZ, M.A., 11; 37

EQUILS, O. see McGREGOR, J.A. et al.

ERB, H. see INFERNUSO, T. et al.

FOWDEN, A.L. see OUSEY, J.C. et al.

FUBINI, S. see INFERNUSO, T. et al.

GILES, R.C. see WILLIAMS, N.M. et al.

GLEED, R. see INFERNUSO, T. et al.

GRAINGER, L. see OUSEY, J.C. et al.

GRAVETT, M.G., 17; 32

HANSEN, P.J. see LEBLANC, M.M.et al.

HARRIS, M. see McGREGOR, J.A.and HARRIS, M.

HARRISON, L.R. see WILLIAMS,N.M. et al.

HONG, C.B. see WILLIAMS, N.M. et al.

HOPPER, R. see CHRISTIANSEN, D.et al.; RYAN, P. et al.

HOUGHTON, E. see OUSEY, J.C. et al.

HUBERT, J.D. see LYLE, S.K. et al.

INFERNUSO, T. et al., 16

JACKSON, C.B. see WILLIAMS,N.M. et al.

JOHNSON, J.R. see LYLE, S.K. et al.

LEBLANC, M.M. et al., 12; 53 andsee CALDERWOOD MAYS, M.B.et al.; CHRISTIANSEN, D. et al.;MURCHIE, T.A. et al.; REBELLO,S. et al.; RYAN, P. et al.

LESTER, G.D. see LEBLANC, M.M.et al.; SANCHEZ, L.C. andLESTER, G.D.

LUZNAR, S. see MURCHIE, T.A. et al.

LYLE, S.K. et al., 15

MACPHERSON, M.L. seeCALDERWOOD MAYS, M.B. et al.; LEBLANC, M.M. et al.;MURCHIE, T.A. et al.; REBELLO,S. et al.

McGLOTHLIN, J.A. see LEBLANC,M.M. et al.

McGREGOR, J.A. et al., 31 andMcGREGOR, J.A. and HARRIS,M., 42

MOULTON, K. see RYAN, P. et al.

MURCHIE, T.A. et al., 33 and seeREBELLO, S. et al.

OLSEN, G. see CHRISTIANSEN, D.et al.

OUSEY, J.C. et al., 38

PACCAMONTI, D., 3 and seeLEBLANC, M.M. et al.; LYLE,S.K. et al.

PALMER, J.E., 7; PALMER, J.E. et al., 45 and see BENTZ, A. et al.

PALMER, L. see OUSEY, J.C. et al.

PELTIER, M.R., 18

POONACHA, K.B. see WILLIAMS,N.M. et al.

READ, R. see RYAN, P. et al.

REBELLO, S. et al., 34

ROSSDALE, P.D. see OUSEY, J.C. et al.

RYAN, P. et al., 13; 35 and seeCHRISTIANSEN, D. et al.

SANCHEZ, L.C. and LESTER, G.D.,48

SCHLAFER, D.H., 6 and seeINFERNUSO, T. et al.

SERTICH, P.L. see BENTZ, A. et al.;PALMER, J.E. et al.

SHEERIN, P. see LEBLANC, M.M. et al.

SMITH, J. see CHRISTIANSEN, D. et al.

STAWICKI, R.J. see LEBLANC,M.M. et al.

SYKES, D. see RYAN, P. et al.

VICKERS, M.L. see WILLIAMS,N.M. et al.

VICKROY, T.W. see MURCHIE, T.A.et al.; REBELLO, S. et al.

VOLKMANN, D. see INFERNUSO,T. et al.

WILKINS, P.A. see BENTZ, A. et al.;PALMER, J.E. et al.

WILLARD, S. see RYAN, P. et al.

WILLIAMS, N.M. et al., 23

WILSON, M. see McGREGOR, J.A.et al.

WITKIN, S. see McGREGOR, J.A. et al.

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Monograph Series No. 19 - Uterine Infections in Mares and Women - A Comparative Study II

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