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EQUINE
INTERNAL
MEDICINE
This page intentionally left blank

EQUINE
INTERNAL
MEDICINE
FOURTH EDITION
Stephen M. Reed, DVM, Dipl ACVIM

Rood & Riddle Equine Hospital • Lexington, Kentucky
Professor Emeritus, Department of Veterinary Clinical Sciences • The Ohio
State University • Columbus, Ohio
Warwick M. Bayly, BVSc, MS, PhD, Dipl ACVIM

Professor, Department of Veterinary Clinical Sciences
Washington State University • Pullman, Washington
Debra C. Sellon, DVM, PhD, Dipl ACVIM

Professor, Department of Veterinary Clinical Sciences • College of Veterinary Medicine,
Washington State University • Pullman, Washington
3251 Riverport Lane
St. Louis, Missouri
63043
EQUINE INTERNAL MEDICINE ISBN: 978-0-323-44329-6
Copyright © 2018, Elsevier Inc. All rights reserved.

Previous editions copyrighted 2010, 2005, 1999.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical,
including photocopying, recording, or any information storage and retrieval system, without permission in writing
from the publisher. Details on how to seek permission, further information about the Publisher’s permissions
policies, and our arrangements with organizations such as the Copyright Clearance Center and the Copyright
Licensing Agency can be found at our website: www.elsevier.com/permissions.
This book and the individual contributions contained in it are protected under copyright by the Publisher (other
than as may be noted herein).
Notices
Knowledge and best practice in this field are constantly changing. As new research and experience broaden
our understanding, changes in research methods, professional practices, or medical treatment may become
necessary.
Practitioners and researchers must always rely on their own experience and knowledge in evaluating and
using any information, methods, compounds, or experiments described herein. In using such information or
methods they should be mindful of their own safety and the safety of others, including parties for whom they
have a professional responsibility.
With respect to any drug or pharmaceutical products identified, readers are advised to check the most
current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be
administered, to verify the recommended dose or formula, the method and duration of administration, and
contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of
their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and
to take all appropriate safety precautions.
To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors assume any
liability for any injury and/or damage to persons or property as a matter of product liability, negligence or
otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the
material herein.
Library of Congress Cataloging-in-Publication Data
Names: Reed, Stephen M., editor. | Bayly, Warwick M., editor. | Sellon, Debra
C., editor.
Title: Equine internal medicine / [edited by] Stephen M. Reed, Warwick M.
Bayly, Debra C. Sellon.
Other titles: Equine internal medicine (Reed)
Description: Fourth edition. | St. Louis, Missouri : Elsevier, [2018] |
Includes bibliographical references and index.
Identifiers: LCCN 2017028516| ISBN 9780323443296 (hardback : alk. paper) |
ISBN 9780323443098 (Ebook)
Subjects: LCSH: Horses--Diseases. | Veterinary internal medicine. | MESH:
Horse Diseases
Classification: LCC SF951 .E565 2018 | NLM SF 951 | DDC 636.1/089--dc23 LC record available at
https://lccn.loc.gov/2017028516
Senior Content Strategist: Jennifer Flynn-Briggs

Content Development Manager: Luke Held
Associate Content Development Specialist: Anna Miller
Publishing Services Manager: Deepthi Unni
Project Manager: Radhika Sivalingam
Design Direction: Amy Buxton
Printed in United States of America
Last digit is the print number: 9 8 7 6 5 4 3 2 1

Contributors
Monica Aleman, MVZ Cert, PhD, DACVIM Katarzyna Dembek, DVM, PhD, DACVIM
Medicine and Epidemiology Assistant Clinical Professor
University of California Veterinary Clinical Sciences
Davis, California Iowa State University
Ames, Iowa
Warwick M. Bayly, BVSc, MS, PhD, DACVIM
Professor Thomas J. Divers, DVM, DACVIM, DVECCS
Department of Veterinary Clinical Sciences Professor of Medicine
Washington State University Clinical Sciences
Pullman, Washington Cornell University
Ithaca, New York
Michelle Henry Barton, DVM, PhD, DACVIM
Fuller E. Callaway Endowed Chair Bettina Dunkel, DVM, PhD, DACVIM, DECEIM,
Large Animal Medicine Dip ACVECC, FHEA, MRCVS
University of Georgia Senior Lecturer in Equine Medicine
Athens, Georgia Veterinary Clinical Sciences
The Royal Veterinary College
Etta Agan Bradecamp, DVM, DACT, DABVP North Mymms
Theriogenologist Hatfield, UK
Rood and Riddle Equine Hospital
Lexington, Kentucky Katherine S. Garrett, DVM, DACVS
Rood and Riddle Equine Hospital
Teresa Ann Burns, DVM, MS, PhD, DACVIM Lexington, Kentucky
Veterinary Clinical Sciences
Clinical Assistant Professor, Equine Internal Medicine Ray Geor, BVSc, MVSc, PhD, DACVIM
The Ohio State University College of Veterinary Medicine Professor and Pro Vice-Chancellor College of Sciences
Columbus, Ohio Massey University
Palmerston North, New Zealand
Jennifer L. Davis, DVM, MS, PhD
Associate Professor of Clinical Pharmacology Tiffany L. Hall, DVM, DACVIM, DACVECC
Department of Biomedical Sciences and Pathobiology Internal Medicine and Critical Care
Virginia-Maryland College of Veterinary Medicine Associate
Blacksburg, Virginia Equine Medical Center of Ocala
Ocala, Florida
Elizabeth Davis, DVM, PhD, DACVIM
Associate Professor Rachel C. Hector, DVM
Clinical Sciences Resident, Anesthesia and Pain Management
Kansas State University Clinical Sciences
Manhattan, Kansas Colorado State University
Fort Collins, Colorado
Igor F. Canisso, DVM, MSc, PhD, DACT, DECAR
Assistant Professor of Theriogenology
Department of Veterinary Clinical Medicine
College of Veterinary Medicine
University of Illinois Urbana-Champaign
Urbana, IL
v
vi CONTRIBUTORS
Kenneth W. Hinchcliff, BVSc, MS, PhD, DACVIM Francisco J. Mendoza, DVM, PhD, MSc, DECEIM
Journal of Veterinary Internal Medicine Professor of Internal Medicine
Co-Editor-in-Chief Department of Animal Medicine and Surgery
Faculty of Veterinary Science College of Veterinary Medicine
Professor University of Cordoba, Spain
University of Melbourne
Melbourne, Victoria Yvette S. Nout-Lomas, DVM, PhD, DACVIM, DACVECC
Australia Assistant Professor
President and CEO College of Veterinary Medicine and Biomedical Sciences
Trinity College Colorado State University
Parkville, Victoria Fort Collins, Colorado
Australia
Alejandro Perez-Ecija, DVM, MS, PhD, DECVP
Melissa T. Hines, DVM, PhD, DACVIM Associate Professor of Internal Medicine
Professor Department of Animal Medicine and Surgery
Large Animal Clinical Sciences College of Veterinary Medicine
University of Tennessee University of Cordoba, Spain
Knoxville, Tennessee
Ann M. Rashmir-Raven, DVM, MS, DACVS, PGCVE,
Samuel D. Hurcombe, MS, DACVIM, DACVECC FHEA
Associate Professor–Clinical Associate Professor
Cornell Ruffian Equine Specialists Large Animal Clinical Sciences
Cornell University Michigan State University
Elmont, New York East Lansing, Michigan
Mary Lassaline, DVM, PhD, MA, DACVO Stephen M. Reed, DVM, DACVIM
Associate Professor of Clinical Equine Ophthalmology Rood and Riddle Equine Hospital
Surgical and Radiological Sciences Lexington, Kentucky
University of California – Davis Professor Emeritus
Davis, California The Ohio State University,
Columbus, Ohio
Maureen T. Long, DVM, MS, PhD, DACVIM
Associate Professor Chris Sanchez, DVM, PhD, DACVIM
Infectious Diseases and Pathology Associate Professor
University of Florida Department of Large Animal Clinical Sciences
Gainesville, Florida College of Veterinary Medicine, University of Florida
Gainesville, Florida
Khursheed R. Mama, DVM, DACVA
Professor of Anesthesiology Debra C. Sellon, DVM, PhD, DACVIM
Clinical Sciences Professor
Colorado State University Department of Veterinary Clinical Sciences
Fort Collins, Colorado College of Veterinary Medicine
Washington State University
Dianne McFarlane, DVM, PhD, MS, DACVIM, CVSH, Pullman, Washington
OSU
Center of Veterinary Health Sciences Maria R. Schnobrich, VMD, Dip ACT
Professor of Physiological Sciences Leblanc Reproduction Center
Oklahoma State Univeristy Theriogenologist
Stillwater, Oklahoma Rood and Riddle Equine Hospital
Lexington, Kentucky
Harold C. McKenzie III, DVM, MS, DACVIM
Associate Professor Harold C. Schott II, DVM, PhD, DACVIM
Large Animal Clinical Sciences Professor of Internal Medicine
Virginia Maryland College of Veterinary Medicine, Virginia Veterinary Clinical Sciences
Tech Michigan State University,
Blacksburg, Virginia East Lansing, Michigan
Robert H. Mealey, DVM, PhD, DACVIM

Professor
Department of Veterinary Microbiology and Pathology
Washington State University
Pullman, Washington
CONTRIBUTORS vii
Colin C. Schwarzwald, PhD, DACVIM, DECEIM Ramiro E. Toribio, DVM, MS, PhD, DACVIM
Professor of Equine Internal Medicine Professor
Equine Department Veterinary Clinical Sciences
Vetsuisse Faculty The Ohio State University
University of Zurich Columbus, Ohio
Zurich, Switzerland
Stephanie J. Valberg, DVM, PhD, DACVIM
Charlie Scoggin, DVM, MS, DACT Professor and Mary Anne McPhail Dressage Chair in Equine
LeBlanc Reproduction Center Sports Medicine
Associate Veterinarian College of Veterinary Medicine
Rood and Riddle Equine Hospital Michigan State University
Lexington, Kentucky East Lansing, Michigan
Clinical Sciences
Affiliate Faculty Bryan M. Waldridge, DVM, MS, DABVP, DACVIM
Colorado State University Veterinarian
Fort Collings, Colorado Park Equine Hospital at Woodford
Versailles, Kentucky
Sharon J. Spier, DVM, PhD, DACVIM
Professor
Department of Medicine and Epidemiology
University of California
Davis, California
Patricia Talcott, DVM, MS, PhD, DABVT

Professor of Toxicology
Washington Animal Disease Diagnostic Laboratory
Washington State University College of Veterinary Medicine
Pullman, Washington
Veterinary Diagnostic Toxicologist
Washington Animal Disease Diagnostic Laboratory
Pullman, Washington

Preface
T
his is the fourth edition of Equine Internal Medicine. Like its three predecessors, it has been written
and edited with the aim of promoting a clearer comprehension of the principles of medical disease
and/or problem development by focusing on the basic pathophysiologic mechanisms that underlie
the development of various equine diseases. As with previous editions, basic information is presented and
then related to the clinical characteristics of each disease and its therapy and management.
Most of the chapters that appeared in the previous three editions have been updated, and a number
of them have been extensively revised or rewritten. Although the bulk of the chapters address specific
diseases along systems-based lines, we realize that the practitioner is initially confronted with a specific
problem that may have its origin in one or more of the body’s systems. The first section of the book is
therefore devoted to an in-depth discussion of the basic mechanisms by which problems might develop,
and the principles underlying the treatment of many of them. The reader can build on this foundation by
reading about specific disorders in the second section of the book, which is divided into chapters dealing
with problems of a particular body system or of a specific nature.
Many true experts have contributed to this text. Their depth of knowledge about all aspects of equine
internal medicine is encyclopedic and daunting. We are grateful for their efforts and diligence in help-
ing us to produce what we hope will continue to be regarded as the definitive text on medical diseases of
horses. We are indebted to them for their efforts. We trust that they derive a sense of pride from the part
they have played in producing what we hope represents the gold standard in equine medical textbooks.
In these days of progressive globalization of the world’s societies and associated growth in the inter-
national movement of horses for breeding, recreational, and competitive purposes, there also has been
a worldwide increase in expectations relating to the standard of veterinary care and evaluation of sick
horses. The sophistication of specialist training programs and the increased number of equine internists
also taking advantage of postgraduate doctoral opportunities have resulted in a wealth of new informa-
tion and the maturing of an increasingly complex and challenging discipline—equine internal medicine.
The delivery of superior health care and increased client expectations that have been associated with
the growth of this discipline have led to the appearance of extremely well-informed and astute equine
general practitioners everywhere, and specialist equine internists on most continents. More than ever
before, equine internal medicine now stands as an autonomous specialty in the veterinary profession. We
trust that the fourth edition of Equine Internal Medicine will prove to have as much universal appeal and
application as those editions that preceded it.
Finally, we would be remiss if we did not thank the many people at Elsevier for their persistence and
efforts. Penny Rudolph, Lauren Harms, Radhika Sivalingam and Anna Miller in particular deserve our
gratitude. They and many others have assisted in manuscript preparation, correspondence, and all the
other tasks that must be completed to get a book like this into print. Without them and the generosity
of our colleagues, this book would not have been published. We think that everyone’s efforts have been
worthwhile.
Stephen M. Reed, DVM, Dipl ACVIM
Warwick M. Bayly, BVSc, MS, PhD, Dipl ACVIM
Debra C. Sellon, DVM, PhD, Dipl ACVIM
ix

Contents
Y PART 1
Mechanisms of Disease and Principles of Treatment
1 Mechanisms of Disease and Immunity, 3
2 Pharmacologic Principles, 79
3 Recognizing and Treating Pain in Horses, 138
4 Critical Care, 158
5 Internal Medicine and Clinical Nutrition, 191
6 Clinical Epidemiology and Evidence-Based Medicine, 218
7 Clinical Approach to Commonly Encountered Problems, 232
Y PART 2
Disorders of Specific Body Systems
8 Disorders of the Respiratory System, 313
9 Disorders of the Cardiovascular System, 387
10 Disorders of the Musculoskeletal System, 542
11 Disorders of the Neurologic System, 580
12 Disorders of the Gastrointestinal System, 709
13 Disorders of the Liver, 843
14 Disorders of the Urinary System, 888
15 Disorders of the Hematopoietic System, 991
16 Disorders of the Endocrine System, 1029
17 Disorders of the Eye and Vision, 1139
18 Disorders of the Skin, 1159
19 Disorders of the Reproductive Tract, 1217
20 Disorders of Foals, 1365
21 Toxicologic Problems, 1460
Appendix I Aspects of Clinical Relevance in Donkeys, 1513
Index, 1525
xi

PART 1
Mechanisms of Disease and

Principles of Treatment
1

C HA P T E R 1
Mechanisms of Disease
and Immunity
Robert H. Mealey and Maureen T. Long*
nondisrupted skin surfaces. Even though horses inhabit an

The Microbiome environment heavily contaminated with fecal flora, normal
dermal flora in the horse is surprisingly devoid of members
Maureen T. Long of the Enterobacteriaceae.2 Normal inhabitants include mixed
populations of bacteria of species of Acinetobacter, Aerococ-
Dermal and mucosal surfaces provide a life-preserving pro- cus, Aeromonas, Bacillus, Corynebacterium, Flavobacterium,
tective barrier composed of physical, chemical, and microbial Micrococcus, Nocardia, coagulase-negative Staphylococcus,
defenses.1 In the past 5 years, the concept of normal flora has Staphylococcus aureus, Streptomyces, and nonhemolytic Strep-
been broadened to include the whole of “microbiota” that tococcus generae.3 Certain Staphylococcus spp. have been
colonizes skin, gut, and mucosal surfaces. The microbiome associated with skin disease in the horse, and these include
is the science of analyzing the total number of organisms in S. aureus, S. intermedius, and S. hyicus, whereas species such
an ecosystem, which in our case are animals. The practical as S. xylosus and S. sciuri were more associated with normal
application of this science to the clinician is that the microbi- skin. More than 30 species of fungi can inhabit the skin and
ome differs among many niches of the body, varies with indi- Alternaria, Aspergillus, Candida, Fusarium, Rhizopus, and
viduals, and has an interactive role with actual disease states. Trichophyton spp. are commonly present.2 Until recently the
Microbiota is the microbial population within the different presence of Malassezia yeast species has been considered
sites of the body. Commensal bacteria are those living on or pathogenic. Recent fungal culturing of the skin of normal,
within a host in a way such that both derive mutual benefit and healthy horses has confirmed colonization by a species of Mal-
in which interruption of this association results in abnormal assezia yeast that is novel to the horse (tentatively named M.
host development or overt disease. These represent that part equi). Colonized sites include the groin, axilla, and perineal
of the microbiome that has intimately coevolved with the dif- regions.4 To date there are no studies describing the microbi-
ferent niches of the body.1 ome of the skin of horses.
Environment is local, such as within different areas of the
gut, and also external, and these can have either positive or Oral Microbiome
negative effects on the microbiome. For instance, poor ven- The oral and pharyngeal mucosa is richly populated with
tilation in animal housing not only is a direct irritant, but many bacteria, including obligate aerobes, anaerobes, and
changes in pH, metabolism of particulates, and activation of facultative anaerobes.5 By culture, gram-positive and gram-
the immune system will dramatically alter the structure of the negative anaerobes are the predominant flora in the mouth
microbiome. and pharyngeal tonsils of the normal horse, with B. fragilis
Age is also an important factor. Colonization of skin and and Bacteroides spp. most commonly found. Genera consist-
mucosal surfaces occurs at birth and is highly variable in early ing of Fusobacterium spp., Eubacterium spp., Clostridium
life. Changes in management from birth to weaning are accom- spp., Veillonella spp., and Megasphaera spp. are also cul-
panied by changes in diet and husbandry for horses. They create tured. Aerobic and facultative anaerobic populations mostly
a highly dynamic environment that eventually can be used to include S. zooepidemicus, Pasteurella spp., E. coli, Actino-
predict best practices once data on the microbiome in healthy myces spp., and Streptococcus spp. In a 16S metagenomics
horses are obtained in a methodical and scientific manner. analysis of the subgingival sites of the horse, although highly
diverse, many similarities to human and other species were
Skin Microbiome observed.6 The most common bacterial phyla included Gam-
The combination of normal flora and mucosal immunity pro- maproteobacteria, Firmicutes, Bacteroidetes, Betaproteobac-
vides an effective barrier against infectious colonization of teria, Fusobacteria, Actinobacteria, Epsilonproteobacteria,
TM7, Deltaproteobacteria, Synergistetes, GN02, Tenericutes,
Spirochaetes, Chloroflexi, and Alphaproteobacteria. The
* The editors and authors acknowledge and appreciate the contributions of Gammaproteobacteria had the highest population of species,
J. Lindsay Oaks, Thomas R. Klei, D. Paul Lunn, and David W. Horohov as with Moraxella spp. and Pasteurellaceae spp. overrepresent-
previous contributors to this chapter. Some of their original work has been ing the population. In the former, Actinobacillus species and
incorporated into this edition. unclassified Pasteurellaceae were the most common bacteria.
3
4 PART 1 MECHANISMS OF DISEASE AND PRINCIPLES OF TREATMENT
The finding of Actinobacillus species is not unexpected; how- ascending colon and cecum, indicating highly specialized
ever, by comparison these taxa are relatively absent from digestive functions associated within the large intestine itself.16
cat and dog oral metagenomes. Of the highly abundant Yeasts and fungi of the order Mucorales have been identified
Firmicutes, many classes of Bacilli were detected; several in the cecum of normal horses and are capable of digesting cel-
Neisseria-like bacteria were detected. Of particular interest lulose and starch.18 By deep sequencing, Firmicutes and Bac-
is the population of oral spirochetes that reside in the equine teroidetes (or in another study, Verruccomicrobia) are the two
mouth because of its relationship to periodontal disease in must abundant phyla.19 The common bovine rumen bacteria
humans, and although there were Treponema, spirochetes, Ruminococcus flavefaciens is one of the most predominant
and leptospires present and abundant, “periodontopatho- cellulolytic bacteria of the equine cecum based on standard
gens” were present in low numbers. When healthy horses microbiologic techniques.15
were compared with horses with oral disease, Actinobacillus Since 2012 several studies have been published investi-
(Gammaproteobacteria) and the Neisseria-like Gemella sp. gating the equine microbiome, and these have been recently
(Firmicutes) predominated compared with Prevotella (Bac- reviewed.17 The techniques employed in each investigation
teroidetes) and Veillonella (Firmicutes).7 could highly bias study results. Fecal samples or samples from
specific gastrointestinal locations have been used. Most stud-
Pharyngeal and Respiratory Microbiome ies had a stated goal of attempting to define the flora of the
Because these same genera are also consistently found in hindgut of the horse.
horses with lower respiratory infections, opportunistic coloni- Routine surveillance demonstrates a relative lack of intesti-
zation by pharyngeal flora is the likely mechanism of disease.5 nal pathogens in the flora of normal horses. In the largest study
Contamination of the trachea of the horse is a frequent occur- to date, fecal shedding of Salmonella enteriditis as detected by
rence, as evidenced by the fact that transtracheal aspiration fecal culture in normal horses from farms without evidence of
yields positive bacterial cultures in approximately 30% of both salmonellosis was 0.8% in resident horses.20 Molecular diag-
normal adult horses and foals.8 As with skin flora, normal nostics, once hoped to provide a tool for understanding the
horses have multiple fungal species inhabiting conjunctival, incidence of Salmonella spp. in the clinically normal horse,
nasal, and oral mucosa.8 Stabling increases the frequency of has provided inconsistent information, and polymerase chain
ocular fungi in normal horses.9 reaction (PCR) is most useful for identification of subclinical
shedders and environmental contamination during an out-
Intestinal Microbiome break.21,22 On the basis of limited investigations, the carriage
In animal models and chronic human conditions, the pres- rates of C. difficile in normal horses and foals appear to be low
ence of normal flora is considered important for intestinal (less than 1.5%).23
maturity and containment of disease. Changes in cecal weight, Intestinal flora in the horse is an important source for
villus:crypt ratio, and volatile fatty acid (VFA) production and extraintestinal pathogens. In studies examining the car-
the development of gut immunoglobulin A (IgA) responses riage rate of Rhodococcus equi, all horses cultured carried
are all affected by suboptimal cecal colonization in germ-free the bacteria regardless of age.24,25 If a farm had endemic R.
animals.10 A relationship between severity of mucosal disease equi and respiratory isolates contain the 90-kDa plasmid
and normal flora has also been demonstrated in models of that is associated with disease, fecal isolates also contained
inflammatory bowel disease of humans.11 this plasmid. In a recent study, there was little difference
Bacteria are present in all parts of the intestinal tract of in the composition of the phyla of fecal bacteria measured
the horse, and the microbial fauna increases in complexity via deep sequencing between normal foals and those with
and density aborally.12 The stomach of the horse is not a ster- either subclinical or clinical R. equi.26 However, a striking
ile environment. A dense population of gram-positive bacte- difference in flora was observed over the first few weeks
rial rods, primarily composed of Lactobacillus spp., colonizes of life among all foals demonstrating the transition from
the nonsquamous portion of the equine stomach. Substantial a juvenile gut to that of the adult. Flora transitioned from
colonization of the duodenum is present with a large popula- equal abundance of Firmicutes to Bacteroidetes over this
tion of proteolytic bacteria, and this colonization increases by short interval.
tenfold in the ileum.13 The right dorsal colon contains the highest numbers
Microbial degradation and fermentation of plant material and the most diversity of protozoal species.17 Four classes
in the large intestine are important components of nutrient of protozoa species, Rhizopoda, Mastigophora, Cliata, and
acquisition in the equid. The consumption of cellulose and Suctoria, have been described based on relative prevalence
starch results in the production of VFAs.14 The major cellulo- in ascending and descending parts of the large colon. The
lytic bacterial strains in the horse produce arrays of fermenta- importance of protozoa for normal gastrointestinal func-
tion products that differ from those of cattle.15 Early genetic tion is debated, but these organisms are presumed to play
techniques also demonstrate that the predominant flora are the an important role in degradation of plant fiber. Despite this
low guanine-cytosine (GC)-content bacteria, which include presumption, one investigation demonstrated little effect of
Cytophaga-Flexibacter-Bacteroides and Clostridium bacteria; protozoa on the digestibility of dry matter with little effect
the actual species are completely novel.16 Standard microbio- on cellulose digestion.27
logic techniques specifically demonstrate Enterobacteriaceae,
Butyrivibrio spp., Streptococcus spp., Bacteroides spp., Lactoba- Urogenital Microbiome
cillus spp., Selenomonas spp., Eubacterium spp., Propionibacte- By far, most of the work that characterizes equine normal
rium spp., and Staphylococcus spp. in residence.17 In addition, flora has focused on urogenital flora to address infertility and
there are completely different compositions of bacteria among fetal loss. Although vaginal and vestibular mucosa of mares
the differing segments of the colon, especially between the should be colonized with normal mucosal flora, the uterus
CHAPTER 1 Mechanisms of Disease and Immunity 5
is considered sterile. However, typical culturing techniques receptor agonist resting in contraction of antral and duodenal
result in frequent isolation of what could be considered patho- smooth muscles.38 In the horse erythromycin results in a dose-
gens, and cytology and bacterial counts are essential supple- dependent increase in ileocecal emptying.39 Motility-enhanc-
mental tests for detecting true uterine infection. Counts less ing effects have also been observed in human patients treated
than 10 colony-forming units and lack of inflammatory cells with amoxicillin.37
indicate uterine or technical contamination.28 Occurrence of infectious lower respiratory disease in adult
Many bacteria inhabit the external genitalia of stallions, horses is an example of how contamination of a normally
including bacteria considered to be associated with metritis sterile site with several commensal bacteria results in disease.
in mares. The predominant aerobe isolated is coagulase- The tonsillar mucosa of the oropharynx is heavily colonized
negative Staphylococcus spp., followed by Corynebacterium with S. equi subsp. zooepidemicus, and necrosis of this tissue
spp., α-hemolytic Streptococcus spp., and Lactobacillus spp. occurring during viral infection is associated with spread to
Pathogens such as β-hemolytic Streptococcus spp., Pseu- the lower respiratory tract.5 Transport of horses (especially
domonas aeruginosa, and Klebsiella spp. can be frequently for distances greater than 500 miles) is a primary risk factor
found in servicing stallions.29,30 Pregnancy rates appear to for pleuropneumonia as demonstrated in a large retrospective
be the same in mares bred to stallions with P. aeruginosa– study.40 Elevation of the head for an extended period of time
infected semen.31 is likely a contributing factor. Horses normally feed from the
ground for most of the day, and this posture promotes effective
Fungal Microbiome tracheal clearance of inhaled debris and particulate matter.
Essentially the same principles apply regarding normal flora, Pasteurella, Actinobacillus, and Streptococcus spp. are the most
host immunity, and specific virulence factors for the patho- frequent and prolific colonizers of the trachea after prolonged
genesis of fungal infection. Fungal infections can be divided head elevation.41,42
into primary or opportunistic pathogens. True pathogens are Nosocomial infection (health care–associated infections)
less dependent on host status than opportunistic pathogens, is defined by the Centers for Disease Control and Prevention
although even a true pathogen may require some degree of as a localized/systemic condition resulting from an adverse
alteration of normal flora or host immunity to become estab- reaction to the presence of an infectious agent or its toxin.
lished. Long-term antibiotic use, immunosuppression, and There must be no evidence that the infection was present or
compromised organ function (especially involving the pul- incubating at the time of hospital admission.43 Nosocomial
monary or endocrine system) are three primary host factors infections are becoming a major problem for large animal
highly associated with the establishment of opportunistic veterinary teaching and private referral hospitals. Infections
fungal infection. Fungi in particular can adapt to the mam- with Serratia marcescens, Acinetobacter baumannii, S. aureus,
malian environment over a relatively short course in order to methicillin-resistant Staphylococcus spp., Enterococcus spp.,
become established. Establishment usually requires a change and various Salmonella enteritidis serovars have all been
in thermal range, oxygen requirements, and resistance to host reported in association with nosocomial infection in equine
defenses. patients.44,45 Surgical incision infection, joint sepsis, cath-
eter phlebitis, wounds, and diarrhea represent the common
Nosocomial Infections clinical syndromes reported in horses.44-46 When nosocomial
Development of colitis in the horse has been associated with infection involves the acquisition of isolates from the hospital
feed change, antibiotics, surgery, nonsteroidal inflammatory environment, these isolates are more difficult to treat because
drugs, and transport, all events that disrupt flora.32,33 Rapid they frequently undergo high-level antibiotic pressure and
change from a roughage diet to concentrate results in increased attain multiple-drug resistance (MDR). Nosocomially trans-
anaerobes, decreased cellulolytic bacteria, decreased cecal pro- mitted salmonellosis in equine hospital wards is increasingly
tozoa diversity, and decreased pH in the equine cecum.32 Iso- reported, with Salmonella enteritidis serotypes Krefeld, Saint
lation of Clostridium difficile is more likely from horses treated Paul, DT104, and Anatum all demonstrating attainment of
with antibiotics, and clinical disease has been associated with MDR over the course of the outbreak.47,48 Only one study of a
ampicillin, erythromycin, penicillin, and potentiated sulfon- nosocomially transmitted S. enteritidis (serotype Heidelberg)
amide administration in adult horses.34,35 In ponies infected did not demonstrate significant acquisition of MDR over
with Salmonella spp., transport and surgery reactivated infec- time.49
tion and diarrhea, and antibiotics (oxytetracycline) prolonged
shedding but did not induce recrudescence.33 In a case-control
study, use of potentiated sulfonamides was not significantly
associated with the development of diarrhea in hospitalized
Pathogenesis of Bacterial Infections
horses; however, overall antibiotic use was highly associated
with the occurrence of diarrhea.36
Antibiotics disrupt normal gastrointestinal flora and func- The ability of bacteria to gain entry and cause disease results
tion,37 and changes in carbohydrate metabolism are a large from a combination of factors possessed by the agent itself,
intestinal event secondary to reduced microbial reduction environmental conditions, and status of host defenses. Gen-
of carbohydrates to short-chain fatty acids (SCFAs). Because eral mechanisms that are specific to bacteria and enhance
SCFA metabolism and absorption result in fluid and electro- disease are virulence factors that enhance the entry, spread,
lyte absorption, a sudden decrease in SCFA leads to osmotic and damage to host tissues (Table 1.1). Major virulence fac-
diarrhea with an intraluminal accumulation of organic acids, tors are listed for specific equine pathogens. Protein secre-
cations, and carbohydrates. Erythromycin and amoxicillin tion systems (PSSs) are a structurally diverse complex of
directly affect colonic motility.37 Erythromycin is a motilin essential virulence factors for bacteria that allow specialized
interactions among cells.50 These main systems function to surface components recognizing adhesive matrix molecules.53,54
translocate various sized molecules and are important in the Gram-positive organisms possess afimbrial proteins on their
formation of adhesins on attachment to host cells. Fibrillar surfaces that presumably aid in binding to host cells. The
adhesins (FAs) and nonfibrillar adhesins (NFAs) are the most three most commonly studied afibrillar adhesins are those
important PSS subgroup not used for bacterial conjugation. that bind salivary glycoprotein, bind fibronectin, or are com-
They specifically target host cells and biofilms for enhance- posed of lipoteichoic acid.53,55 Salivary binding proteins are
ment of colonization and invasion. There are multiple types commonly found in pathogens and commensals of the oral
of FAs in both gram-positive and gram-negative bacteria, cavity. Both Streptococcus spp. and Actinomyces spp. possess
with gram-negative types the most well characterized (Table these proteins. Fibronectin binding protein (FBP) is neces-
1.2).51,52 Pili or fimbriae are rod-shaped structures composed sary for S. aureus invasion and binds both fibronectin and
of an orderly array of a single protein usually arranged in collagen to form a bridge between the FBP and the host cell
a helical fashion to form a cylinder. The tip of the fimbria integrin (integrin α5β1).56,57 Heterologs of FBP have been
mediates attachment to carbohydrate moieties on cell sur- demonstrated in S. pneumoniae of humans, S. equi subsp.
faces and is integral to bacterial invasion and colonization. equi, and S. equi subsp. zooepidemicus. Other potential equine
Bacteria can also contain multiple types of pili. Both the bac- pathogens that have FBPs on their surface include Actino-
terial pili themselves and the cellular pathways they use for myces spp., E. fecalis, and L. monocytogenes.55,58 Lipoteichoic
secretion and formation of pili are targets for pharmacologic acid, a common binding factor found in Streptococcus group
intervention, and there are multiple subclasses depending on A bacteria, is important in adhesion of bacteria to cells.59 This
their configuration.51 protein is also important in stimulation of cytokine secretion
Afimbrial adhesins are cell proteins that enhance the bind- from the cells during infection and has been demonstrated in
ing of bacteria to host cells. They are also called microbial group B Streptococcus, including S. equi subsp. equi.60 A less
commonly described afibrillar adhesin is composed of surface
TABLE 1.1 General Mechanisms of Bacterial Pathogenesis polypeptide chains in Corynebacterium that binds to lectin.61
Afibrillar adhesins are also present in gram-negative organ-
Action Mechanism Examples isms; the most commonly studied are conserved high-molec-
Entry of bacteria Adhesion Fibrillar adhesins ular-weight adhesion proteins of Haemophilus influenzae and
Entry Nonfibrillar adhesins Bordetella pertussis.59
Curli fimbriae Bacterial ecology has emphasized the importance of bio-
Lipoteichoic acid film for colonization of both biotic and abiotic surfaces of
Biofilm adhesion proteins bacteria.50 A requirement for biofilm formation is a tight
Membrane ruffling interaction of bacterial adhesins with surface receptors that
promote further bacterial aggregation. The most important
Enhancement of Immune resis- Capsule
family of biofilm adherence proteins is that of Staphylococ-
spread tance Lipopolysaccharide
cus aureus. These proteins have high molecular mass and
Host sub- Anticomplement
repetitive structures whose size and number can be varied
strate Resistance to
during the course of infection, possibly allowing for immune
utilization phagocytosis
evasion.
Phagolysosomal survival
The most common virulence proteins of both gram-pos-
Iron acquisition
itive and gram-negative organisms are bacteria lectins.53-62
Damage to host Toxins Exotoxin Although attachment is thought to be the primary role of
membranes Endotoxin these proteins, attachment itself results in an intracellular
Apoptosis change, including actin rearrangements, cell signaling reg-

ulation, or actual secretion of bacterial substances into the
TABLE 1.2 Major Types of Bacterial Adhesins

Adhesin Definition Example
Type 1 fimbriae Cell surface structure primarily on gram-negative bacteria that bind Escherichia coli
to the terminal mannose of glycoproteins on cells
Type 4 pili Cell surface structure primarily on gram-negative bacteria that func- Pseudomonas aeruginosa
tion in adhesion, twitching, and DNA uptake, which bind on CD46
and other glycolipids
Curli fimbriae Coiled aggregative fimbrial structures that bind to fibronectin, lam- Enterohemorrhagic Escherichia
inin, and plasminogen and function in adhesion, aggregation, and coli (EHEC)
biofilm formation Salmonella
Fibrils and flexible rods Short, thin rodlike adhesins that bind to fibronectin for adhesion to Streptococcus species
host tissues
Lipoteichoic acid Part of the peptidoglycan layer of cell walls Gram-positive bacteria
Biofilm Exopolysaccharide produced by bacteria that allows matrix forma- Gram-positive and gram-
tion of embedded material negative bacteria

host cell. These proteins are highly conserved in bacteria and in vitro phagocytosis can be abolished with treatment with
are important targets for immunoprophylaxis. Membrane hyaluronidase and induction of specific immunity against
transformation for uptake of intracellular bacteria such as M-protein of S. equi subsp. equi and S. equi subsp. zooepi-
Yersinia spp., Listeria monocytogenes, Salmonella spp., and demicus.80 The M-proteins of Streptococcus spp. are also essen-
Shigella flexneri can be either zipperlike or triggerlike. Alter- tial for resistance to phagocytosis blocking complement.66,81-83
natively, Salmonella and Shigella bacteria adhere and secrete This mechanism for complement resistance appears to be
proteins that are translocated into the host cell cytoplasm through enhancement of binding of fibrinogen to the bacteria
and trigger actin polymerization.63 In addition to mem- in the presence of M-protein.79,84,85
brane ruffling, Mycobacterium avium and Salmonella spp. Apoptosis is a distinctive morphologic process that results
also rely on activation of intracellular GTPases, leading to in cleavage of nuclear material and scavenging of unwarranted
phagocytosis.64,65 cells without immune activation. Apoptosis or programmed
Once colonization occurs, multiplication and spread of cell death is an important pathway for complex organisms
bacteria are enhanced through virulence factors. These fac- to deal with damaged and diseased tissue. Apoptosis avoids
tors assist bacteria in survival in the hostile host environ- the release of the tissue-damaging enzymes and nonspecific
ment and breakdown of tissue barriers. One of the most elimination of tissue that occurs in cellular necrosis. Several
common and potent strategies for avoidance of phagocytosis bacteria have modulated the host apoptotic pathways for
is the presence of a capsule. Despite the remarkable diver- enhancement of survival.86 Shigella flexneri, S. typhimurium,
sity of bacteria, capsule assembly and structure are remark- and toxins of S. aureus, Pseudomonas spp., and C. diphtheriae
ably similar across species. Early studies with S. equi subsp. have demonstrated programmed cell death as a consequence
equi demonstrated that resistance to phagocytosis was asso- of cellular infection or exposure.87,88 The protein of S. flex-
ciated with an increase in capsule and M-protein,66 and in neri, IpaB, induces apoptosis by binding to and activating the
a model of S. equi subsp. zooepidemicus infection in mice, cellular enzyme caspase 1, which induces apoptosis of mac-
enhancement of virulence was associated with increased rophages.89 Staphylococcus aureus α-toxin, which is similar
amount of capsule, which increased resistance to phagocy- to listeriolysin O, presumably escapes the macrophage after
tosis.67 Although colonization of the guttural pouch occurs engulfment and induces host cell apoptosis.90 The TSST toxin
with nonencapsulated S. equi subsp. equi strains, induction of S. aureus induces B-cell apoptosis and blocks immunoglob-
of lymphadenopathy is associated with capsular strains.68 In ulin production.91
more recent studies of S. equi subsp. equi infection, rapid
colonization in the lingual and pharyngeal tonsil is depen-
dent on genetically associated virulence factors that control
colony morphology, with the mucoid strain having enhanced
Pathogenesis of Fungal Infections
virulence.69 Maureen T. Long
Capsules of anaerobic bacteria are unique, and these
structures may directly account for the formation of Of the 250,000 species of fungi, fewer than 200 are true
abscesses within the host. The capsule of B. fragilis has two pathogens.1 Superficial mycoses affect the hair shaft and the
distinct polysaccharides composed of repeating subunits superficial epidermis. Cutaneous mycoses (dermatophytosis)
with oppositely charged groups (Zwitter ion).70 This poly- infect the epidermis, dermis, hair, and nails of animals, and
saccharide complex injected alone promotes the induction Microsporum, Trichophyton, and Epidermophyton spp. are
of abscess. Infection of rodents with the encapsulated form the most commonly associated pathogenic genera. Subcuta-
of Bacteroides and Fusobacterium spp. results in the forma- neous tissues can become infected with Sporothrix, Conid-
tion of intraperitoneal abscesses, whereas nonencapsulated iobolus, Basidiobolus spp., and members of the Dematiaceae
bacteria do not cause abscessation.71-73 Synergism of capsu- fungi, including the Chromoblastomycosis, Mycetoma, and
lar anaerobes with other bacteria occurs; nonencapsulated Phaeohyphomycosis spp. infections. Most of these infections
bacteria have enhanced survival in abscesses and produce are introduced by penetration through skin or opportunistic
capsule when cultured or inoculated with encapsulated invasion of damaged skin surfaces. Histoplasma capsulatum,
bacteria.72 Coccidioides immitis, Blastomyces dermatitidis, and Para-
Similar to and overlapping with capsule are structural pro- coccidioides brasiliensis are the four most important fungal
teins that block complement. The O side chain of lipopolysac- pathogens that can cause systemic infection. The most com-
charide (LPS) on gram-negative bacteria is an anticomplement mon opportunistic infections include Candida albicans,
factor.74 The longer the side chain, the greater the distance Aspergillus spp., Cryptococcus neoformans, Mucor spp., and
between phagocytes and bacteria. The capsular component, Pneumocystis carinii.
sialic acid, interacts with O antigen to prevent the formation Fungal virulence factors may be more complex than those
of C3 convertase.75 Bacterial enzymes are formed by Strepto- of bacteria because of the higher degree of opportunism that
coccus spp. and other organisms that damage the polymorph occurs with a change in host status. There may be subtle fac-
chemoattractant, C5a.76,77 Production of a protein in Salmo- tors that, combined with host status, result in a certain fungus
nella spp., encoded by the rck gene, prevents insertion of the attaining a virulent state. For instance, the typical fungal wall
C9 fragment of complement into the bacterial membrane.78 is composed of three major polysaccharides: mannose; β-1,3
The M-protein of S. equi subsp. equi appears to decrease depo- and β-1,6 linked glucans; and chitin. Chitin mutants in C.
sition of complement on the surface of bacteria.79 albicans are less virulent when tested in rodent models than
Recent studies of streptococci have shown that when are wild-type fungi.92 Further, a mutant C. albicans that can-
M-protein content is kept constant, the amount of capsule not synthesize complex mannose oligosaccharides does not
is correlated with resistance to phagocytosis.68 Resistance to adhere to other yeast and epithelial cells and has lost virulence
in a guinea pig model.93 Both of these mutants can proliferate

in vitro normally, and whether or not this is an actual viru- Pathogenesis of Viral Infections
lence factor is unclear.
As with bacteria, cellular adherence is an important pre-
requisite for infection and colonization of the host. Adhes-
ins have been identified in C. albicans and B. dermatitidis. Viral infections such as equine influenza (Orthomyxoviri-
Two genes have been associated with adhesion in C. albi- dae), rhinopneumonitis and abortion (Herpesviridae), Afri-
cans. The first is a glycoprotein that has sequences consis- can horse sickness (Reoviridae), equine infectious anemia
tent with agglutinating activity. Transformation of this gene (Retroviridae), and various encephalitides (Alphaviridae
into other nonadherent fungal species results in adhesion and Flaviviridae) are responsible for some of the most medi-
of the transformed yeast to cells.94 Candida albicans also cally and economically important diseases of horses. Specific
has integrin-like proteins, the disruption of which results therapy of viral infections remains a significant challenge
in diminished hyphal growth, adhesion to cells, and loss because antiviral drugs are generally ineffective, impractical,
of virulence in mice.95,96 The B. dermatitidis adhesin medi- or prohibitively expensive for the treatment of horses. Treat-
ates binding to human monocyte-macrophages through the ment of most viral infections focuses on supportive care of
CD14 receptor.97 the affected organ systems and control of secondary compli-
Many fungi have polysaccharide capsules that, like bac- cations such as bacterial infection. Currently control of most
teria, help resist phagocytosis and immune activation. The clinically significant viral diseases in horse populations relies
capsule of C. neoformans inhibits leukocyte accumula- on vaccination, quarantine, or even destruction of infected
tion, cytokine secretion, and macrophage phagocytosis.98 animals.
Mutants without capsule are highly infective and avirulent. Despite the great significance of some viral infections,
As indicated earlier, many fungi are engulfed by macro- recognizing that many equine viruses are ubiquitous,
phages, and intracellular survival is mediated by virulence weakly pathogenic, or not associated with any known dis-
factors. Macrophages are decimating cells for C. albicans,99 ease under normal circumstances is also important. Some
H. capsulatum,100 and B. dermatitidis. Histoplasma cap- examples include equine adenovirus, respiratory and
sulatum is primarily a yeast in vivo, and this form infects enteric reoviruses (the term reo is derived from the acro-
macrophages. Phagolysosomal fusion occurs at a normal nym for respiratory enteric orphan, indicating that these
rate,101 but blockage of acidification of the phagolysosome isolates have not been associated with disease), and equine
occurs.102 herpesvirus (EHV) type 2. Some host-virus relationships
Exposure to both pathogenic and saprophytic fungi is an may be mutualistic in that virally derived genetic elements
everyday occurrence. Respiratory contamination and infec- are theorized to benefit the host by facilitating genetic
tion are important for many pulmonary species, but skin pen- variability and evolution.116 Thus many viruses are of no
etration and dissemination from necrotic gut are important practical clinical significance, and no control efforts are
portals for large animals also. Dissemination after the initial warranted. For this reason, the veterinarian should never
infection is dependent on previous damage to host tissues, assume that the recovery of a virus from a clinical specimen
deeper mechanical penetration, or actual invasion of new tis- is significant without proof that the virus can cause the dis-
sues. C. albicans can actually grow through and replace cell ease in question.
membranes.99 True molds invade blood vessels and grow An in-depth discussion of viral structure, taxonomy, and
along the intima of the vessels. Fungi secrete many degradative replication is beyond the scope of this chapter, and the reader
enzymes, including proteinases, phosphatases, and DNAses, is referred to textbooks of veterinary or human virology for
to surmount structural barriers.103 A group of genes called more detailed information.117,118 A brief overview is presented
secreted aspartyl proteinase (SAP) genes allow more persistent to emphasize those features that have clinical relevance.
colonization of host surfaces and deeper penetration.104 The fundamental structure of all viruses is a DNA or RNA
When C. immitis invades the host, the fungi form endo- genome enclosed by a coat of protein called the capsid (Fig.
spores. These endospores secrete a proteinase and a urease 1.1). For viruses that are enveloped, the capsid is enclosed
that likely aid in the breakdown of pulmonary tissues.105-107 further by a host cell–derived lipid membrane into which
The two proteinases of A. fumigatus break down elastin, a viral proteins have been incorporated. In addition to protect-
major component of lung tissues.108,109 Phospholipase activity ing the viral genome, the capsid and other associated struc-
has been demonstrated in C. albicans, C. neoformans, and A. tural proteins (e.g., matrix proteins) are important for virus
fumigatus.110 Strains of Candida spp. with high amounts of this assembly, packaging the viral genome, releasing the genome
enzyme have higher virulence,111 and abolishing this activity into a target cell, and for nonenveloped viruses providing
results in decreased adherence of the organism.112 Host eico- receptors that bind to host cells. For enveloped viruses the
sanoids enhance fungal colonization. Recent evidence dem- receptors are incorporated into the lipid membrane. The pri-
onstrates production of eicosanoids by both dermatophytosis mary clinical significance of these features is that enveloped
and systemic fungi.113 viruses, because of their fragile lipid membrane, are highly
Fungi induce apoptosis, which may be due either to the susceptible to inactivation by heat, desiccation, or deter-
direct effect of a fungal toxin or secondary to host cell cyto- gents, and transmission typically requires direct exchange of
skeleton rearrangements.114 The gliotoxin of A. fumigatus can body fluids, short distance aerosols, or arthropod vectors. In
induce DNA fragmentation and apoptosis in macrophages.115 contrast, nonenveloped viruses (e.g., equine rotavirus) are
This toxin also has many other immunosuppressive qualities, resistant to physical inactivation, and environmental con-
which include inhibition of the neutrophil respiratory burst tamination is more likely to be a significant factor in their
and T-cell activation. transmission.
Herpesvirus Lipid envelope mutations are neutral or even deleterious, in the face of selective
(host cell derived) pressures such as the host immune response or antiviral drugs,
Capsid this genetic plasticity allows rapid development of resistant
(icosahedral) virus populations.120 Secondary structures or certain sequences
Genome in the viral genome may facilitate polymerase errors further at
Tegument
selected regions that are important for immune evasion, such as
in sequences that code for neutralizing epitopes.119 The genome
Surface viral of some viruses, such as influenza, comprise separate segments,
glycoproteins which allows reassortment of entire gene segments and sudden
(with receptors) and dramatic changes in antigenicity.121
All viruses are obligate intracellular parasites. Viral replica-
tion can occur only within living cells, and all viruses to some
extent depend on the host cell synthetic machinery. The life cycle
Rotavirus of all viruses includes the following steps: attachment to the tar-
get cell, entry into the cell, uncoating and release of the viral
genome, transcription and translation of viral proteins, replica-
Genome
tion of the viral genome, assembly of new virions, and release
(11 segments) of progeny virions117,118 (Fig. 1.4). Although the biochemistry of
these steps is beyond the scope of this discussion, recognizing
Capsid that they are all specific, energy-requiring interactions between
(2 layers of protein) the virus and the host cell is important. The inability of the virus
to interact appropriately with a cell at any of these steps prevents
Outer capsid proteins replication in that cell type and defines the tropism of the virus.
(with receptors) All of these steps are also important potential targets for antivi-
ral drugs and host immune responses.
Once the viral nucleocapsid gains entry to the cytosol of
FIG. 1.1 Schematic representations of basic viral structure. The basic struc- the cell, the viral genome is released through the process of
ture of an enveloped virus is shown by the drawing of a herpesvirus. The ba-
uncoating. After uncoating, the viral genome localizes to the
sic structure of a nonenveloped virus is shown by the drawing of a rotavirus.
appropriate regions of the cell for replication and mRNA tran-
scription. DNA viruses typically replicate genomes and tran-
scribe mRNA in the nucleus and then transport the mRNA to
the cytoplasm for translation. RNA viruses typically replicate,
The composition of the viral genome is an important basis transcribe mRNA, and translate viral proteins in the cyto-
for virus classification (Fig. 1.2). The type of viral genome also plasm. These sites of replication account, respectively, for the
determines the strategies required to replicate the genome and location of viral inclusion bodies that are diagnostically useful
transcribe messenger ribonucleic acid (mRNA; Fig. 1.3). Viral in histopathologic sections.
genomes may be single-stranded RNA, double-stranded RNA, Cells that replicate virus often are killed as a direct con-
single-stranded deoxyribonucleic acid (DNA), or double- sequence of infection. One mechanism by which viruses kill
stranded DNA. The genomes of single-stranded RNA viruses cells is lysis, often associated with the release of progeny viri-
may have positive polarity, in which the genome also serves ons. Insertion of viral proteins into cell membranes, budding,
directly as mRNA for translation of viral proteins. To replicate direct toxicity of viral proteins, and diversion of normal host
genomes, these viruses must first synthesize a strand of com- cell homeostatic processes to viral replication may result in
plementary RNA that can be used as a template to replicate death of the cell.117,118,122 Viruses also may activate the cellular
genomes and transcribe new mRNA. Retroviruses are a subset self-destruct mechanism of programmed cell death (apopto-
of single-stranded, positive-polarity RNA viruses that use their sis). Although cells may induce apoptosis in an attempt to pre-
RNA genomes as templates to produce double-stranded DNA, vent completion of the virus life cycle, viruses also may use this
which in turn is used for the transcription of mRNA and new mechanism to kill the cell and facilitate release of virions.123
viral genomes. Single-stranded RNA viruses may also have Viral infection can cause neoplastic transformation of
negative polarity, in which the genome is antisense to mRNA, infected cells. The most common examples in horses include
and synthesis of a complementary RNA strand is required to warts (equine papillomavirus) and sarcoids (bovine papillo-
serve as mRNA and as a template for new genomes. The clini- mavirus). Virally induced invasive neoplastic diseases such
cal significance of these types of replication strategies is that as leukemia or lymphosarcoma have not been recognized in
they require polymerases and other enzymes not normally the horse. Viral proteins that activate the cell cycle into the
found in eukaryotic host cells. These unique viral enzymes are growth and division phases may lead to neoplastic transfor-
important targets for antiviral drugs because they can be used mation if expressed in a cell that is not killed by the infection.
selectively to inhibit viral replication. Papillomavirus infections induce epithelial neoplasms (fibro-
Viral RNA polymerases are low fidelity and lack proofread- papillomata) using a virally encoded protein (E5 oncoprotein)
ing functions and thus randomly introduce errors into new that induces proliferation of normally quiescent cells and that
RNA at an average rate of about one nucleotide mismatch per presumably is needed for viral replication.124 Oncogenic retro-
10,000 bases copied.119 In a population of viruses, therefore, viruses, including leukemia and sarcoma viruses, induce neo-
virtually every individual virus differs slightly, and this popula- plastic transformation by integration into the host cell genome
tion is referred to as a quasi-species. Although many of these and activation of cellular oncogenes.125
ENVELOPED NONENVELOPED
dsDNA dsDNA
DNA Polyomaviridae
Iridoviridae Adenoviridae
Hepadnaviridae
Herpesviridae ssDNA
Poxviridae, Chordopoxviridae
Parvoviridae
ssRNA dsRNA
Reoviridae
Coronaviridae Paramyxoviridae Bunyaviridae Toroviridae
Birnaviridae
RNA Orthomyxoviridae Arenaviridae Togaviridae Flaviridae ssRNA
Piconaviridae
Retroviridae Rhabdoviridae
Caliviridae
Filoviridae
FIG. 1.2 Virus family classification based on genome composition and presence of envelope or absence
of envelope. The relative size of the viruses to each other is also shown.
At the host level viruses may use several mechanisms to infected animals serve as the reservoir of the virus.127-129 On
establish persistent infections and avoid immune clearance. reactivation viral nucleic acids are translocated across syn-
Virologic latency is defined as the presence of a viral genome apses to epithelial cells of the nasopharynx, which produce
that is not producing infectious virus.126 The genomes of infectious virus. The stimuli that induce reactivation are
latent viruses are transcriptionally suppressed and trans- poorly defined, but reactivation can be induced by immuno-
lationally silent so that no viral proteins are expressed that suppression (e.g., corticosteroids) and presumably by other
may identify the cell to the immune system as infected. The stressors, such as pregnancy, transport, and social stress.128,130
definition of latency also stipulates that on reactivation, viral The severity of disease in a virus-infected horse, or whether
gene expression and the production of infectious progeny infection even results in clinical disease at all, is the result
virions can be resumed, differentiating latently infected cells of a complex interaction among the triad of virus, host, and
from cells infected with defective viruses. In contrast, some environment. Critical factors include viral virulence, viral
persistent viral infections are characterized by continual rep- spread within the animal, the intensity of direct and immune-
lication despite the presence of antiviral immune responses. mediated pathologic response elicited by the virus, and the
Even in the absence of recognizable clinical signs, such infec- ability of the virus to avoid clearance by the host. Other than
tions are not truly latent. The classic latent infection is that the virulence of the virus, which is strictly a property of the
of the herpesviruses. For the α-herpesviruses, such as EHV1 virus, the other virus-host interactions can be influenced by
and EHV4, latent infections are established in the nuclei of the age and genetics of the host and by environmental fac-
sensory neurons and can be maintained indefinitely, and tors such as stress and nutrition. These factors account for
Protein Synthetic Replicative New Viral

Expression Intermediates Viral Genome Intermediates Genome
dsDNA
Adenoviridae
Asfarviridae
Herpesviridae
(+) mRNA Iridoviridae dsDNA
Papillomaviridae
Polyomaviridae
Poxviridae
ssDNA
(+) mRNA dsDNA Circoviridae (– or +) dsDNA ssDNA
Parvoviridae (– or +)
dsRNA
(+) mRNA Birnaviridae dsRNA
Reoviridae
ssRNA (+)
Arteriviridae
Astroviridae
Caliciviridae
(+) mRNA ssRNA (–) Coronaviridae ssRNA (–) ssRNA (+)
Flaviviridae
Picornaviridae
Togaviridae
dsDNA Retroviridae dsDNA
ssRNA (–)
Arenaviridae
Bornaviridae
Bunyaviridae
(+) mRNA Filoviridae ssRNA (+) ssRNA (–)
Orthomyxoviridae
Paramyxoviridae
Rhabdoviridae
FIG. 1.3 Summary of the main virus families that infect vertebrates and general strategies employed by
these viruses to produce mRNA for protein expression and to replicate genomes. Required intermediate mol-
ecules are indicated. White arrows indicate the need for unique viral polymerases, including RNA-dependent
RNA polymerase and RNA-dependent DNA polymerase (reverse transcriptase). Dark arrows indicate the use
of cellular polymerases or viral homologs of cellular polymerases. ds, Double-stranded; ss, single-stranded;
(+) for RNA = positive polarity, polarity of RNA used for protein translation; (+) for DNA = coding strand,
sequence same as for (+) RNA; (− or +) DNA = contains single strands of DNA of both polarities. (Modified
from Baltimore D: Expression of animal virus genomes, Bacteriol Rev. 35:235-241, 1971.)
the observation that considerable variation in disease signs in virulence, presumably because of the greater number of
can occur among a group of animals infected with the same infected cells and amount of tissue damage. The virulence
viral strain. of equine infectious anemia virus strains can be correlated
Certain strains of a virus may cause more severe disease directly to plasma virus titers and numbers of infected cells,
than other strains. The main properties of a virus that may without any changes in tropism.131,132 The molecular basis
affect virulence include host cell tropism and replication for the increased replication rate is not clear but most likely
rate. A tropism change that leads to involvement of addi- is caused by variation in viral regulatory sequences and
tional tissues or facilitates virus spread generally results in proteins.133,134
more severe disease. Outbreaks of EHV1 abortion or neu- Viral infections generally are regarded as localized or sys-
rologic disease strongly suggest that EHV1 strains exist temic. Localized viral infections are those that are restricted
that have a tropism for these tissues compared with EHV1 to a single organ system, often at the site of entry. Because
strains that cause respiratory disease. An increase in the infection of the tissue is direct, the incubation period for
viral replication rate is usually associated with an increase localized viral infections is usually short, often only a few
1 (Fig. 1.5). Localized viral replication first occurs at the site of

entry and in regional lymph nodes. Depending on the level
of replication, clinical disease may be present. The virus then
Cytoplasm enters the blood or lymphatics and spreads to other tissues,
2
such as spleen and liver, in which clinical disease may occur.
Virus is amplified and then released again for a second, usually
9 higher-titered, viremia that further disseminates the virus to
3 Nucleus
other organs. Each viremic episode is associated with a febrile
response and is the basis for the biphasic fever response asso-
4 6 ciated with some viral infections. Because systemic infections
6 4
require multiple steps, the incubation periods are longer than
5 for localized infections, typically 1 to several weeks. Infec-
10 tions in the horse by eastern, western, or Venezuelan equine
encephalitis virus closely follow this paradigm. Localized viral
7
7 11 replication occurs at the site of entry (mosquito bite) followed
by viremia and dissemination to the central nervous system.139
8 For most horses, even nonvaccinated horses, dissemination is
8 controlled before infection of the brain, and neurologic dis-
ease is a rare outcome of infection. A variation on the theme
is infection of horses with EHV1. The most common clini-
cal disease associated with EHV1 infection is rhinopneumo-
FIG. 1.4 Schematic representation of the general virus life cycle. Both nitis caused by a localized infection of the nasopharyngeal
RNA and DNA viruses share the initial steps, including attachment (1); entry/
mucosa.140 In almost all cases, a cell-associated viremia also
fusion (2); and uncoating and release of viral genome into the cell (3). Solid
occurs in lymphocytes, but in most infected horses this does
arrows indicate remaining steps for RNA viruses, which all occur in the cyto-
not result in disease. However, in some cases, viremia is asso-
plasm, including transcription of mRNA (4); translation of viral proteins (5);
ciated with infection of endothelial cells, and in the pregnant
replication of the viral genome (6); assembly of new virions (7); and release
mare vascular damage to the uterus and placenta may lead to
of progeny virions (8). Dashed arrows indicate steps for a DNA virus. The life
abortion.140,141 Similarly, infection of the vascular endothelium
cycle is similar except that the DNA genome is translocated to the nucleus (9)
in the central nervous system results in neurologic disease.142
for transcription (4) and replication of viral genomes (6). Viral mRNA is then
Some viruses also may spread in the host through nerves.
translocated back to the cytoplasm for translation (10), and newly synthesized
In the horse rabies is the best-known infection that relies on
viral proteins are translocated back to the nucleus (11) for assembly (7); new
neural spread. Following local replication in myocytes at the
virions are then released from both nuclear and cytoplasmic membranes (8).
site of entry, usually a bite wound, rabies virus ascends periph-
eral nerves into the central nervous system, where it repli-
cates in neurons and then egresses by way of cranial nerves
days. Many infections of the skin or mucosal surfaces are to the salivary gland.143 EHV1 and EHV4 establish latency in
localized, and examples in the horse include infections with the nuclei of sensory neurons that innervate the nasopharynx
enteric rotavirus and influenza. For influenza, virus is inhaled and reach the nucleus by ascending nerve axons. Similarly, on
into the nasopharynx and replicates in epithelial cells of the reactivation these viruses egress back down the axon to infect
upper respiratory tract and trachea. Virus is not present in epithelial cells.127,144
the blood or tissues outside of the respiratory tract. In gen- Once a virus reaches a target organ, virally mediated cell
eral, viruses remain localized because they lack the recep- death is the fundamental source of pathologic response,
tors to infect cells of other tissues or circulating cells, such as disease, and clinical signs observed by the veterinarian.
monocytes or lymphocytes, that can disseminate the virus. Despite the great complexity of virus-host interactions
Some viruses are temperature sensitive and remain local- and the many factors that influence the expression of clini-
ized because they are unable to replicate efficiently at core cal disease, in actuality viruses have a limited number of
body temperatures. EHV3, the cause of coital exanthema, is ways by which to cause pathology. Cells and tissues may be
restricted to the surface of the genitalia in horses because of destroyed directly by cytolytic viral infections or by infec-
its temperature sensitivity.135 EHV1 and EHV4 are not tem- tions that affect the differentiated function of target cells
perature sensitive, however, and systemic infection may occur (e.g., neoplasms and immunodeficiencies). Viral infections
with these viruses. Temperature sensitivity is also a means of organ systems with bacterial flora (e.g., intestinal and
by which some viruses, such as equine influenza and infec- respiratory tracts) can disrupt the normal barrier functions
tious bovine rhinotracheitis virus, may be attenuated for use of these organs and result in secondary bacterial infections
as modified-live intranasal vaccines. Infection by the vaccine and toxemia that may contribute significantly to the patho-
strain is limited to the cooler mucosal surfaces; the inability logic response. Cell death and pathologic response also may
to spread systemically prevents sequelae such as abortion and be caused by host immune responses specifically directed
pneumonia.136,137 against virally infected cells or by indiscriminate inflam-
Systemic infections are those in which virus is dissemi- matory responses. Virally induced autoimmune diseases
nated to multiple tissues by blood or lymph. This viremia may have not been described in horses but are another potential
exist in the form of cell-free virions in the plasma or lymph source of pathologic response that may be identified in the
or may be cell associated in circulating blood cells, usually future.
monocytes or lymphocytes. The classic paradigm for a sys- For most of the clinically important viral diseases of horses,
temic infection is infection of mice with ectromelia virus138 disease manifestation results from some combination of
DAY
Skin: Invasion
Multiplication
1 Regional lymph node:

Multiplication
2
Bloodstream:
Primary viremia
3
Incubation Spleen and liver:
period Multiplication necrosis
4
Bloodstream:
5 Viremia
Skin: Focal infection

6 Multiplication
Swelling of foot
(Primary lesion)
8
Early rash
Papules
Disease 9
10
Severe rash
Ulceration
11
FIG. 1.5 Schematic representation of the pathogenesis of mousepox (ectromelia), illustrating the classic
paradigm for the events in a systemic infection. (From Fenner F: The pathogenesis of the acute exanthems,
Lancet. 252:915, 1948.)
cytolytic infection and immune-mediated tissue destruction. apparent. EHV1 infections of the respiratory epithelium,
The relative contribution of these mechanisms is primarily a which has a large number of cells with a high turnover rate,
function of viral virulence and host factors that influence the produce mild clinical disease even with a high rate of infec-
type and intensity of immune responses. The predominant tion. On the other hand, much more significant clinical
mechanism of pathologic response also can vary with differ- manifestations of EHV1 infection such as abortion and neu-
ent stages of the same disease, as seen in acute versus chronic rologic disease are caused by infection of a few endothelial
equine infectious anemia. In acute disease most of the disease cells because minimal vascular damage can lead to thrombo-
manifestation is caused by direct viral damage and cytokines, sis, ischemic necrosis, and damage to large amounts of tissue.
whereas in chronic disease immune complex–mediated ane- If viral infection results in neoplastic transformation of a cell
mia and glomerulonephritis become more significant. type, disease may progress according to the characteristics of
The disease associated with a given viral infection is the neoplasm, whether or not the virus remains associated
related to the affected organ system(s), the number of cells with the tumor.
destroyed, and the sensitivity of the affected organ system to In most viral infections immune-mediated pathologic
dysfunction. If the number of infected cells is not sufficient response contributes significantly to disease and in some
to lead to clinically significant organ dysfunction, the result cases may be the predominant cause of disease manifestation.
is a subclinical infection. When enough cells are infected to Equine immune and inflammatory responses are covered else-
lead to overt organ dysfunction, clinical disease becomes where in this chapter.
Although not described in horses, viral infections in other in disease conditions. Immune responses leading to protective
species can induce immune-mediated responses to host cell resistance against reinfection occur, but the level of this resis-
antigens and autoimmune diseases. The best documented tance is most often incomplete. Mechanisms associated with
human autoimmune disease suspected to be initiated by viral these responses have not been investigated extensively in the
infections is Guillain-Barré syndrome, in which infection with horse, but information is available from other host-parasite
cytomegalovirus or Epstein-Barr herpesvirus elicits antinerve systems that may be relevant to equids. The purpose of this
ganglioside immune responses and demyelinating disease.145 section is to acquaint the reader with contemporary thoughts
Postinfluenza myocarditis is an occasional sequela in horses on host-parasite interactions. Because of their prevalence,
and human beings and is a potential autoimmune disease. major importance to equids, and information available, cover-
Although no direct evidence supports this theory, influenza age is limited to helminth parasites that occur in most devel-
virus is not identified consistently in affected heart muscle, oped and nontropical countries.
and the pathogenesis is not known.146 Infection with most metazoan parasites results in inflam-
A key requirement for viruses to be maintained in nature mation and structural and functional changes of the organs
is to persist successfully in a reservoir host (if the reservoir is invaded. The outcome of these changes is an alteration of the
an infected animal) and to be transmitted to another suscep- host’s physiologic state. The degree of alteration depends on
tible host. One of the most important obstacles to persistence the existing physiologic condition of the animal, which is dic-
and transmission is detection and elimination by the host tated to a great degree by its age, nutritional status, and previ-
immune system. Within the host rapidly replicating viruses ous immunologic experience with the parasite. The numbers
such as influenza may shed and transmit virus before the host of parasites introduced and the specific parasite also affect the
can mount specific antiviral immune responses. Herpesvi- degree of physiologic change that occurs. When these fac-
ruses avoid detection during latency by not expressing any tors favor major alterations, the results are readily identifiable
viral proteins. Immunodeficiency viruses may cripple antivi- clinical signs of infection. Subclinical infections, although less
ral immune responses by directly infecting immunoregulatory apparent, are potentially important to the general health of the
CD4+ T lymphocytes. One of the most important mechanisms animal and continued transmission of the agent. The patho-
of immunologic avoidance is antigenic variation in which neu- physiologic effects of infection by ectoparasites, helminths, and
tralizing viral antigens are altered so that they are no longer microorganisms are in many cases similar.150,151 Abnormalities
recognized or accessible by host immune responses. Antigenic in weight gain, skeletal growth, reproduction, and lactation may
variation is generated by nucleotide errors during transcrip- result from infections with any of these agents. These changes
tion or replication, which result in amino acid substitutions are often directly related to parasite-induced anorexia, disrup-
in the relevant epitopes. Other mechanisms by which some tion of metabolic processes, and anemia. An understanding of
viruses may modify their antigenicity is through intramolecu- the morphologic and biochemical lesions produced by specific
lar recombination/duplication or reassortment of segmented parasites clarifies the role of these agents in clinical and sub-
genomes (e.g., influenza and African horse sickness).120,121 For clinical conditions associated with the infections. Most detailed
reassortment, coinfection of a single cell with genetically dif- studies of the pathophysiology of parasitic infections have been
ferent virions may result in a progeny virion with segments conducted in laboratory animal models and domestic animal
derived from both virions and a major change in antigenicity. species other than the horse.151 However, the classical pathol-
In influenza these are called antigenic shifts, and the radical ogy of parasitic infections of the horse has been reviewed.149,152
change in the antigenicity of the virus may render preexisting The following discussion outlines some recent observations on
immunity in the host population ineffective at preventing out- host-parasite interactions that may be of significance to equine
breaks of disease with high morbidity and mortality rates.121 medicine. Examples of host-parasite interactions responsible
Genetic differences in susceptibility to disease have been for alterations in host homeostasis are presented as they relate
well documented. In an outbred population of animals, the to the gastrointestinal tract, lungs, and skin.
considerable variation in the type or severity of clinical dis-
ease is well recognized, even when animals are infected with Y GASTROINTESTINAL TRACT
the same virus strain and have no recognizable differences
in other factors such as age, challenge dose, nutrition, and Internal parasites are most important to equine health as
general health status. Conversely, highly inbred populations mediators of gastrointestinal problems, including colic and
may be more uniformly susceptible to a viral disease.147 Thus diarrhea. Although almost all internal parasites have been
inbreeding can pose problems for endangered species, such as inferentially implicated as causative agents of colic at some
Przewalski’s horse, or other populations with limited genetic time, significant evidence-based experimental or field obser-
variability, which may incur high rates of morbidity or mortal- vations have emerged to support this contention for some
ity if the animals are infected with a virulent virus. Host genet- parasites. The helminth parasites include large strongyles,
ics may affect the tropism of the virus and influence the type principally Strongylus vulgaris, Parascaris spp., Anoplocephala
and intensity of immune responses to a viral infection. perfoliata, and as a group the cyathostomins.
The pathogenesis of colic associated with migration of
Strongylus vulgaris through the mesenteric arteries and the
resultant thrombosis, infarctions, and necrosis of the intes-
Pathogenesis of Parasitic Infections tine have been described in detail elsewhere.149,152,153 Large
strongyles are easily controlled with currently available mac-
rocyclic lactone anthelmintics and are rare in horses kept on
Horses serve as hosts for numerous parasites, which induce well-managed farms in developed countries. Histologic stud-
a wide range of pathologic and immunologic responses.148,149 ies of experimentally infected parasite-free pony foals dur-
Many of the latter are of a hypersensitive nature and also result ing the initial stages of infection indicate that the severity of
lesions produced in the intestine cannot be attributed solely ruptures and intussusceptions of the cecum and colon asso-
to mechanical disruption caused by larval migrations and that ciated with these infections.160,161 Several case-control studies
these larval stages induce some biologic amplification system have documented an association between colics in the ileal
within the mucosa, which results in the degree of inflamma- region and A. perfoliata infection.162 These parasites inhabit
tion observed.154 Although the mechanisms involved in this the region of the ileocecal junction and produce ulcerated
response have not been investigated, the histologic nature of lesions of the mucosa and submucosal inflammation. Detailed
the lesion is characteristic of an Arthus reaction, suggesting experimental investigations of these infections have not been
an involvement of the immune response. Other experimental conducted, and thus specific details on the pathogenesis and
studies using the parasite-free pony–S. vulgaris system have relevance of these lesions are lacking. An association between
implicated a role for the immune response in the mediation severity of observed lesions and tapeworm burden has been
and regulation of the arterial lesions produced by this para- observed.163,164
site. Passive transfer of immune serum but not normal serum Cyathostomins (cyathostomes or small strongyles) have
reduced the severity of arteritis seen and clinical signs asso- not generally been considered to be of major importance,
ciated with experimental infections without reducing the particularly as causative agents of colic. In this regard,
numbers of parasites that develop in these ponies.155 However, Uhlinger’s field studies are of particular importance.165,166 In
treatment with immune serum also induced an anamnestic these controlled experiments different anthelmintic treatment
eosinophilia and marked perivascular infiltration of eosino- regimens were used to test their efficacy in reducing the inci-
phils in the cecum. The reduction in intravascular lesions may dence of colic. The more efficacious treatment programs sig-
have been associated with an inactivation of parasite-secreted nificantly reduced the incidence of colic by 2 to 13 times that
inflammatory factors either by antibody or serum enzymes seen in the same herds before implementation of the more
or circulating cytokines. This serum may also have contained efficacious treatment. Because of the management programs
nonspecific host-derived antiinflammatory substances. The used before the initiation of this study and the results of fecal
exacerbation of the eosinophil response may have been asso- cultures, it can be assumed that the primary parasites pres-
ciated with the formation of immune complexes. Although the ent in these horses were cyathostomins. These data strongly
mechanisms are unknown, the results suggest that the immune implicate a role for cyathostomins in a substantial proportion
response may simultaneously modulate and potentiate inflam- of colics observed under field conditions. The parasite or host
mation. It has been postulated that larvicidal treatment of S. factors involved in these colic cases are unknown but may be
vulgaris–infected horses and killing of intravascular larvae the dynamic turnover of parasites during the life cycle in the
may release a bolus of antigenic factors from these larvae mucosa and the related inflammatory responses seen that they
within the mesenteric vasculature, resulting in an exacerba- induce.
tion of arterial and intestinal lesions and colic. Experimen- Cyathostomins have been implicated in numerous case
tal testing of this hypothesis indicates that this phenomenon reports with seasonal diarrhea in adult horses, which is a con-
does not occur and further that viable larvae are necessary to dition called larval cyathostominiasis. These cases are charac-
maintain the arteritis and eosinophilia seen.156 Experimental terized by a sudden onset of diarrhea during the late winter
studies using parasite-free ponies that were immunized with or spring. Horses younger than 5 years of age are particularly
crude adult worm antigens and subsequently challenged with at risk, but mature horses can be affected as well, and a case-
S. vulgaris larvae showed an exacerbation of the pathologic fatality rate of 50% has been reported.167 These are difficult
responses seen in the mesenteric vasculature and including to diagnose, and the only consistent signs are weight loss,
an anamnestic eosinophilia, further suggesting a role for the hypoproteinemia, and diarrhea.166 Large numbers of larval
immune response in the development of these lesions.157 cyathostomins can be found in the feces or in intestinal con-
Colic associated with Parascaris spp. infection in foals has tents and within the mucosa of these horses. These symptoms
been related to intestinal impaction and rupture and is not are related to the synchronous emergence of fourth-stage
considered to be of major significance in adult horses.158 These larvae of these parasites from the mucosa. These larvae build
conditions in foals may become more important with the to potentially large numbers within the mucosa owing to the
widespread identification of Parascaris spp. resistance to iver- arrested development of infective larvae. The seasonality of
mectin.159 However, ascarid nematodes are particularly potent the occurrence of this condition at present does not appear
sources of allergens, and it is not inconceivable that the hyper- to vary in different climatic regions as does the analogous
sensitized mature horse may respond to low-level infections bovine condition of type II ostertagiasis. Specific parasite or
with this parasite. Observations made in the author’s labora- host factors associated with the regulation of the hypobiotic
tory are noteworthy in this regard. Two mature Parascaris-free state of the larvae or the inflammatory response initiated at
adult horses were inoculated intradermally with less than 90 parasite emergence have not been described. Other clinical
μg of saline-soluble somatic extract of adult Parascaris spp. reports have suggested that cyathostomin-related diarrhea
to test for immediate hypersensitivity to this antigen. Both and weight loss are not related to the seasonal presentation
horses experienced an immediate systemic response and colic. described previously.168 This is supported by experimental
One of the horses died within 3 hours of intradermal inocula- studies demonstrating that pathophysiologic effects occur as
tion. Necropsy results were consistent with the diagnosis of large numbers of parasites enter the intestine, as well as when
acute severe colitis. Because of the allergic potential of ascarid they leave.169
nematodes and the sensitivity of the equine gut to immediate In view of the paucity of specific mechanistic information
hypersensitivity reactions, this potential is worthy of further on the pathophysiologic effects of equine gastrointestinal par-
characterization and consideration. asites, a synopsis of relevant information gathered from other
Clinical observations have maintained an interest and model systems is warranted, particularly for nematodes.151,170
concern over the pathogenic potential of Anoplocephala per- Parasitic organisms may induce changes in gastrointestinal
foliata infections. Earlier case reports have described cecal function directly by mechanical disruption of tissues and cells
or by the release of factors that directly alter cell function. Parascaris spp. larval migrations in the lungs of yearling
Induction of the immune response serves as an anamnestic horses produce more severe clinical signs and inflammatory
amplification system. The result of these changes is an altera- responses than in foals that are reared parasite free. These
tion in function of the smooth muscle and epithelium of the infections in yearlings are accompanied by focal accumula-
bowel. tions of lymphoid tissue, indicating an induction of an active
A number of helminth parasites, including Parascaris local immune response. It is suggested that this is an age-
spp., stimulate intestinal smooth muscle hyperplasia. This related phenomenon.178 However, it is likely that more severe
response may be induced by intestinal inflammation or ste- reactions could be the result of previous sensitization to Par-
nosis associated with parasitism. Contractility of these mus- ascaris spp. antigens. Increased responses of this nature have
cles can be induced in a Schultz-Dale reaction by stimulation been described in the livers of pigs immunized with Ascaris
with parasite antigens. This response is mediated in rats suum antigens following challenge infections.
by mast-cell–derived 5-hydroxytryptamine (5-HT) and in Dictyocalus arnfieldi infections of donkeys rarely produce
guinea pigs by histamine. A regulatory relationship of myen- clinical signs, and it has been suggested that these equids are
teric neurons to these antigen-induced changes has also the natural host for this parasite. Infections of horses produce a
been demonstrated in this model system. These latter experi- more severe and prolonged bronchial inflammatory response
ments suggest that antigen-induced stimulation of smooth similar to Dictyocalus sp. infection in other hosts. The mecha-
muscle contractility may be correspondingly blocked by nisms associated with this differential response have not been
γ-aminobutyric acid similarly stimulated by mast cell prod- defined but are not uncommon in unadapted host-parasite
ucts. This complex system may be an adaptation by the host associations. It is possible that the more marked inflammatory
to maintain homeostasis in the face of continued antigenic reaction of the horse to these parasites is due to the absence of
stimulus. It is noteworthy that strongyle-induced altera- downregulatory mechanisms that are established in the more
tions in myoelectric activity of the equine small intestine and adapted natural host, the donkey.
colon have been demonstrated in vivo.171,172 In some of these
experiments, dead S. vulgaris larvae evoked an alteration of Y SKIN
the smooth muscle response in previously exposed ponies,
suggesting a role for the immune response in the stimulation Whereas Onchocerca cervicalis infections in horses are rare
of the hyperactivity.173 where macrocyclic lactone anthelmintics are regularly used,
There is a rapidly growing literature on the host-parasite reactions to the filarial nematode illustrate variations seen in
interactions of a number of mouse models of gastrointestinal responses to chronic parasite infection. Focal, alopecic, depig-
nematode infections.174-177 The mouse models include infec- mented, pruritic lesions are often seen in infected horses. Not
tions of Trichinella spiralis, Nippostrongylus brasiliensis, Helig- all infected horses react to this infection, and the appearance
mosomoides polygyrus, and Trichuris muris. Because of the of clinical signs is often seasonal. Detailed studies have not
use of the mouse the most contemporary of immunologic, been conducted on the pathogenesis of these lesions in horses.
genetic, molecular, and cell biologic techniques are available However, similar conditions occur in human onchocercia-
and may provide useful insights into potential explanations of sis,179 and it is likely that the host-parasite responses active in
immunologic and pathophysiologic responses of the horse to humans are also present in the horse. Lesion development is
nematode parasites, particularly the cyathostomins. Although associated with immune-mediated killing of microfilariae in
differences in specifics exist among these murine models, it is the skin. Parasites appear to be killed in an antibody-depen-
clear that these worms induce a type 2 T-helper cell cytokine dent cell-mediated reaction. In this response antimicrofilar-
response consisting of interleukins (ILs) IL-4, IL-5, IL-9, and ial surface IgG and IgE antibodies mediated adherence and
IL-13. IL-4 and IL-13 induction of the Stat6 pathway is cen- degranulation of granulocytes, which are predominantly
tral to most of the resulting responses. There is a consistent eosinophils. Major basic protein of eosinophils has been dem-
mastocytosis and eosinophil infiltration of the intestine. The onstrated in the tissues of patients with dermal lesions, and
resulting responses also involve goblet cells, the enteric ner- it has been suggested that eosinophil toxic enzymes and pro-
vous system, epithelial cells, and alternately activated macro- teins are responsible for many of the changes seen. The rea-
phages as effector cells. Cell junctions are disrupted, smooth son for the absence of these lesions in most horses is unclear.
muscle hypercontractility is stimulated, epithelial cell secre- Human onchocerciasis and filariasis are spectral diseases.
tion increased, and goblet cell production of mucus is stim- In these diseases regulation of immune responses has been
ulated. Although these mechanisms are used most often to associated with the lack of pathologic responses to the para-
explain expulsion of the nematodes in question, they could be sites.180,181 Immune regulatory mechanisms associated with
equally important in the disruption of the homeostasis seen in these infections include immune tolerance, anergy, induction
the parasitized equine intestine. of immune regulation involving Treg cells or macrophages,
and the production of IL-10 and transforming growth factor
Y RESPIRATORY SYSTEM beta (TGF-β)–altering Th-cell subsets and the production of
specific cytokines during different phases of the infections.
Several nematode parasites infect the equine lung. These Recent studies have also focused on the role of an intracellular
include migrating stages of Strongyloides westeri and Paras- commensal microorganism, Wolbachia, which is a parasite of
caris spp. en route to the small intestine. Migrating stages of Onchocerca and other filarial nematodes. These bacteria have
aberrant parasites, such as Habronema sp., Draschia megas- been shown to mediate type 1 inflammatory lesions in humans
toma, and Strongylus spp., which induce granulomatous foci in and mouse models.180
the lung parenchyma, and adults and larvae of the lungworm The presence of these types of parasite-associated immune
Dictyocalus arnfieldi, which inhabit the bronchi, also occur. regulatory events has yet to be critically studied in the horse.
Host responses to two of these are noted. However, the seasonal variability in skin responses to the
Onchocerca microfilariae by horses in some regions has been the migrating L3, which is effective in preventing reestablish-
investigated. In this instance the onset of ventral-midline der- ment of the intestinal infection but is ineffective against L3,
matitis during the summer may be related to a seasonal fluc- which are sequestered in the abdominal fat of the sow.184 Simi-
tuation in microfilarial burdens of the skin, which have been lar epidemiologic phenomena occur in S. westeri infection of
demonstrated to correspondingly peak at this time.182 Not only horses, and it may be implied that similar immunologic mech-
do total numbers increase, but microfilariae are also found anisms are also active.
more commonly in the surface layers of the skin. Interestingly, Immunologic mechanisms associated with protective resis-
this period of abundant microfilariae corresponds with the tance are presented primarily as they relate to parasites that
seasonal peak in numbers of the vector, Culicoides varripennis. inhabit the lumen of the gastrointestinal tract and secondarily
Although speculative, correlations in the peak availability of as those that undergo extraintestinal tissue migration.
microfilariae and vectors may be an evolutionary adaptation Immune responses directed toward gastrointestinal nema-
by these parasites to maximize transmission and survival of todes vary significantly among hosts and against different para-
this parasite species. site species within a given host. However, some generalities may
serve as a background for understanding these responses in the
Y PROTECTIVE RESISTANCE horse. A phenomenon termed self-cure has been described in
sheep, in which the ingestion of significant numbers of infec-
Resistance to infection may be innate or acquired. In some tive larvae induces the expulsion of existing adult parasites.
instances innate resistance to equine parasites has been attrib- This expulsion is initiated by a species-specific immediate-type
uted to age, with older individuals being resistant. Most equine hypersensitivity response that may cause the nonspecific expul-
helminth parasites develop only in the horse, and conversely sion of other nematode species. Although this phenomenon
the horse exhibits an innate resistance to most nonequine has not been examined in the horse, experimental infections of
parasites. Exceptions to this rule are parasites with a broader naturally parasitized ponies with large numbers of S. vulgaris
host range that occasionally infect horses, such as larvae of the L3 induced a dramatic decrease in preexisting strongyle fecal
tapeworm Echinococcus granulosus and the liver fluke Fasciola egg counts, suggesting that a self-cure–like reaction may occur
hepatica. Trichostrongylus axei, a parasite of ruminants, estab- under some conditions.
lishes readily in the equine stomach and produces significant More typically, establishment of primary infections results
lesions only when present in large numbers. In some cases at some time in spontaneous expulsions of these worms as a
parasites that develop in the horse induce more severe lesions result either of senility or, as demonstrated in laboratory ani-
and clinical signs than in their apparent normal host, as has mal model systems, of active acquired immune responses.
been described for D. arnfieldi. This phenomenon occurs experimentally in the absence of
Age resistance to Parascaris spp. and S. vulgaris has been reinfection and is thus separate from the self-cure phenom-
described in horses by comparing susceptibility of young and enon. A large number of immune effectors have been iden-
old ponies reared under parasite-free conditions. It is apparent tified with this phenomenon in various model systems, and
that the reaction of the lung to migrating Parascaris larvae is it is likely that some if not all are at some time active in the
more marked in mature horses and suggests that an immune equine intestine. The mechanisms involved are T-cell depen-
response occurs in this site.160,161 Initial reports on age-acquired dent. Antibodies may be involved but are not sufficient in
resistance to S. vulgaris infection183 have not been substanti- themselves to induce expulsion. T-cell–mediated mastocy-
ated, and experimental observations in our laboratory indicate tosis, eosinophilia, and goblet cell hyperplasia have all been
that this does not occur. demonstrated to be related to expression of expulsion in some
The occurrence of acquired resistance to equine parasites systems. These accessory cells are involved in the nonspecific
can be inferred from the observation that older chronically efferent arm of this response. Mediators of inflammation, such
exposed horses generally have lower burdens of parasites than as vasoactive amines, prostaglandins, and increased produc-
do similarly exposed young horses. On the basis of these cri- tion of mucus, have been linked to immune elimination of
teria, acquired resistance is apparent to infections of S. westeri, primary infections in some but not all model systems. The
Parascaris spp., Strongylus spp., and cyathostomin species. increased secretion and hyperreactivity of intestinal smooth
Extensive experiments are limited, however, to those on S. muscle associated with worm expulsion have been termed
vulgaris and to some degree cyathostomins. weep and sweep phenomena. It is likely that a number of spe-
Although S. vulgaris has been largely eliminated from well- cific immunologic events initiate several nonspecific effector
managed horse farms, examination of details of the immune mechanisms, resulting in this expulsion. These mechanisms
response to it illustrates some equine immune mechanisms. vary with the species of parasite involved. The elimination of
Resistance that is acquired to S. vulgaris in most cases is partial adult S. westeri and Parascaris spp. from maturing horses and
and of a concomitant type; that is, some stages of the para- the hypothetical seasonal turnover in Strongylus spp. and cya-
site, such as arterial larvae of S. vulgaris, may reside within the thostomin spp. may be mediated by such responses.
horse in the face of an active acquired resistance against newly In addition to immune responses that occur during tissue
acquired infective stages. migrations, protective resistance to reinfection by gastroin-
Resistance to infection with S. westeri adult parasites is testinal nematodes occurs at the surface of the epithelium.
inferred by the short duration of their life cycle within the This reaction, termed rapid expulsion or immune exclusion,
small intestine and the failure of subsequent exposures to is separate from self-cure or immune expulsion of primary
establish patent infections. Mares, however, remain infected infection. Infective larvae are expelled from the intestine in
with arrested third-stage larvae, which subsequent to foaling a matter of hours. Again, mechanisms of expulsion described
are transmitted to the foals in milk starting at 4 days postpar- vary between parasite and host species. However, anaphy-
tum. Similar phenomena occur in swine strongyloidosis. In lactic reactions and mucus entrapment have been observed.
these infections there is apparent protective resistance against Some experiments using the T. spiralis–rat system suggest
that alterations in the epithelial cells in immune animals are eosinophils are essential in this protective immune response,
directly involved in the exclusion of these parasites. Although an anamnestic eosinophilia is characteristic in immune ponies
immune-mediated damage of intestinal helminths such as but not nonimmune ponies following experimental S. vul-
decreased fecundity, reduced size, and morphologic alterations garis challenge.157 Because of its prominence and compelling
have been noted, infective larvae expelled by rapid expulsion in vitro and correlative in vivo data, the eosinophil is consid-
mechanisms remain viable and undamaged. It may be specu- ered a major effector in immune-mediated helminth killing.
lated that reactions of this nature are responsible, in part, for However, recent studies in murine parasite model systems in
resistance to reinfection of equines with cyathostomins. which eosinophilia was blocked by anti–IL-5 treatment sug-
Some specific observations have been made on the equine gest that this type of cell is not essential for protective resis-
acquisition of resistance to reinfection with cyathostomins.185 tance in some systems.190 Significant increases in equine IL-5
This resistance is acquired over time with continued exposure have been measured in ponies vaccinated against S. vulgaris.
to pasture L3; is incomplete; and, like other parasitic helminth It is possible, in vivo, that a number of cells function as effec-
infections, appears to be genetically regulated in that parasite tors and may overcome the absence of sufficient eosinophils
numbers are overdispersed within a given herd of adult ani- under some circumstances. Antibody reactivity with parasite-
mals. The limited number of experimental and field studies secreted enzymes and molting fluids, factors important in
completed to date suggests that immunity may be directed parasite homeostasis, has been demonstrated in vitro; similar
toward all stages of the parasite life cycle. Challenge infections reactions may be important in vivo.
of previously exposed and naive ponies with mixed strongyle T-cell responses are essential for the induction of protec-
infections indicate that a nonspecific self-cure–like phenom- tive resistance to tissue-migrating helminths in most systems
enon occurs, reducing numbers of adult and fourth-stage lar- studied, including the experimental S. vulgaris pony model.
vae (L4) in the lumen as well as within the mucosa.157 Fecal This dependency is likely due to the T-cell dependency of the
egg counts are significantly reduced in older animals com- antibody response and to the mediation of secondary effec-
pared with yearlings even when adult parasite numbers are tor cell responses. It is likely that antigenic substances secreted
similar. These observations suggest that immune responses or excreted (ES) by migrating nematodes are important in the
are directed at female fecundity or that the species of cyathos- induction of these responses. It is probable that a combina-
tomins present in the older animals are inherently less fecund. tion of immune responses elicited by a combination of specific
Numbers of early L3 (EL3) are reduced following challenge parasite antigens, including surface antigens and ES products,
infections of previously exposed adult ponies compared with is necessary to induce an immune response sufficient to pro-
previously exposed young animals or age-matched naive con- vide protective resistance.
trols.186 Other studies have suggested that acquired resistance
is important in the induction of hypobiosis.169,187 Resistance
to acquisition of EL3 was not seen when numbers were com- Y PARASITE-INDUCED REGULATORY
pared in young previously exposed and age-matched ponies RESPONSES
raised under parasite-free conditions. However, these previ-
ously exposed young animals demonstrated significantly fewer Helminth parasites have evolved elaborate mechanisms
developing larvae (DL) in the mucosa, suggesting that once to evade the host’s immune responses, live in the face of
the EL3 began to develop, they were susceptible to immune an active specific host response, and yet establish chronic
attack.186 This response is likely driven by cytokines produced infections. These immune evasion strategies include the
by T cells of the Th2 lineage with demonstrated increases in production and secretion of molecules such as proteases,
IL-4 and IL-5. This response mimics in some ways the results protease inhibitors, antioxidants, prostaglandins, and
described in studies of murine nematodes.177 phosphorylcholine-antigens, which disrupt host effector
A number of intestinal helminths, as well as others, migrate responses.191,192 Other molecules that mimic host immune
through extraintestinal tissues as part of their life cycle. regulatory factors, such as TGF-β and (macrophage migra-
These include parasites such as Parascaris spp., S. westeri, and tion inhibitory factor) MIF, have also been identified in
Strongylus spp., all of which stimulate an acquired immune parasitic helminth genomes.191 These mimics likely down-
response in the horse. During this migration these larvae regulate host-parasite–directed protective response and
are vulnerable to attack by immune effectors that may either add to immune evasion.
encapsulate them in an immune-mediated inflammatory Helminth parasites can induce a regulatory immune
response, disrupt their migrations by interfering with impor- response, which promotes their survival but also has bystander
tant metabolic or invasive processes, or inhibit molting from effects on host responses to other infectious agents, vaccines,
the L3 to L4 stages. The most studied phenomenon in this allergic responses, and autoimmune diseases. These observa-
regard is antibody-mediated adherence of inflammatory cells, tions have progressed to the point that helminth therapy is
which may result in killing of the larvae. This phenomenon being developed for the treatment of human inflammatory
involves many cell types and immunoglobulin isotypes in bowel disease and ulcerative colitis.193,194 In this situation
different host-parasite systems. In vitro studies of this nature infection with the swine whipworm T. suis impedes the regu-
have been conducted using S. vulgaris third-stage larvae and lation of type 1 inflammatory responses responsible for these
equine immune effectors.188 In these experiments an antibody- conditions and reduces disease activity. The generally accepted
dependent adherence of cells was demonstrated and shown to and simple model that has emerged for this type of helminth-
be parasite species specific. In vitro killing was mediated by induced regulation is that parasite factors stimulate dendritic
eosinophils and not by neutrophils or monocytes. Activated cells that activate regulatory T cells and alternately activated
eosinophils were necessary to mediate this response, and S. macrophages. These cells, through the production of TGF-β
vulgaris infections have been demonstrated to activate eosino- and IL-10, may suppress both Th1- and Th2-mediated inflam-
phils and neutrophils in vivo.189 Although it is not certain that matory responses.195-197
Detailed studies of the effects of equine helminths on the infectious diseases, and an important one is pneumonia
regulation of parasite-induced responses or to heterologous caused by Rhodococcus equi. Entry into the host can occur
immunogens have not been reported. The effects of dif- by ingestion, inhalation, or penetration through epithe-
ferent levels of gastrointestinal helminth infection on the lial barriers such as the skin or mucosal surfaces. Regard-
response of ponies to keyhole limpet hemocyanin immu- ing the entry of organisms, inoculum size is a key factor in
nization was measured.198 Antibody levels determined by determining whether or not disease will develop. Follow-
enzyme-linked immunosorbent assay showed that animals ing entry, microorganisms must spread from the entry site
with low levels of parasites had a trend toward increased to local tissues or disseminate to distant sites in the body.
KLH-specific total immunoglobulin, IgG(T), and IgA Bacteria such as Staphylococcus aureus produce enzymes
compared with heavily parasitized ponies. Moderately and such coagulase, protease, and hyaluronidase that facilitate
heavily parasitized ponies demonstrated a trend toward their ability to spread through tissues and cause disease.
reduced lymphoproliferative response to KLH that was not Substances produced by microorganisms that enhance their
restored after the addition of IL-2. Cells from these ponies ability to cause disease, as well as microbial structures that
also produced significantly lower levels of IL-4 compared do the same, are collectively referred to as virulence factors.
with lightly parasitized ponies. These data indicate that The next step in the development of disease is replication of
heavily parasitized animals have uniformly decreased cel- the infectious agent to levels that result in clinical signs. This
lular and humoral immune responses to soluble protein period of replication before the development of clinically
immunization. The mechanisms involved are unknown. A apparent disease comprises the incubation period. Micro-
recent study evaluated the inflammatory and immunologic organisms that cause disease by producing toxins, such as
response to three different antigens given simultaneously botulinum toxin produced by Clostridium botulinum, are an
to ponies concurrently dewormed with either ivermec- exception to this rule. Disease in the host ensues when the
tin or pyrantel pamoate or kept as untreated control. This infection results in tissue damage. There are many ways in
study found no difference in cytokine expression, acute- which damage can occur. Cell death caused by replication of
phase inflammatory markers, or antigen-specific antibody the agent, extracellular toxins produced by the agent, and,
titers between the anthelmintic treatment groups.199 Taken importantly, the host’s inflammatory and adaptive immune
together, the possible interaction between resident intesti- responses directed against the agent all result in tissue
nal nematodes and responses to vaccination in horses is in damage and disease. The systemic inflammatory response
need of further investigation. elicited by endotoxin during gram-negative bacterial infec-
tions is an excellent example of host-mediated damage. The
final outcome depends on the interplay between infectious
agent factors and host factors, which include the inoculum
Infection and Immunity size and virulence of the agent, as well as the immune sta-
Robert H. Mealey tus of the host. The host may mount a successful immune
response and clear the organism, as occurs with equine
influenza virus infection, or the immune response may
Y BASIC PRINCIPLES OF INFECTIOUS control replication of the agent such that disease resolves
DISEASE but the agent is not eliminated. This results in persistent
inapparent infection as occurs with equine infectious ane-
Equine clinicians devote tremendous effort to the preven- mia virus infection. Finally, highly virulent agents such as
tion, diagnosis, and treatment of infectious diseases. Detailed rabies virus and Hendra virus will kill the host, whereas less
and current information on specific infectious diseases in the pathogenic agents may kill hosts that are immunocompro-
context of organ systems is provided elsewhere in this book, mised in some way.
and in-depth discussions of infectious diseases of horses are Successful pathogens have developed efficient mechanisms
provided in a recent text200 and review.201 The field of equine for entry, spread, and replication, all of which enhance their
infectious disease research is growing rapidly, and modern ability to cause disease. Importantly, each one of these steps
genomics techniques such as massively parallel deep sequenc- requires breaching or avoiding host defense mechanisms,
ing have enabled the recent discovery of previously unknown which include physical barriers, innate immune responses, and
equine pathogens and potential pathogens.202,203 In addition, adaptive immune responses. These same immune responses
continued application of proteomics approaches204 will lead to ultimately control most infectious agents but also participate
a better understanding of infectious disease pathogenesis and in the pathogenesis of disease. Thus understanding how infec-
the discovery of novel vaccine and therapeutic targets, as well tious diseases develop and how they are controlled requires a
as improved diagnostics. fundamental knowledge of immunology, which is the primary
On a more basic level, the following events occur in all focus of this chapter.
infectious diseases: encounter, entry, spread, multiplication,
damage, and outcome.205 Encounter occurs when the host Y EQUINE IMMUNOLOGY
comes into contact with an infectious agent. The encounter
is endogenous when infections are caused by normal micro- Although our modern understanding of the immune sys-
bial flora. An example is pneumonia caused by Streptococcus tem is primarily based on humans and rodent models, the
zooepidemicus, a member of the normal flora of the equine horse has contributed significantly to our understanding of
nasopharynx. Exogenously acquired infections result from many immunologic processes. These contributions include
encounter with an infectious agent in the environment the earliest work on serotherapy and passive transfer of
or an agent transmitted from other animals or via insect antibodies, antibody structure and function, immunity to
or tick vectors. There are many examples of exogenous infectious agents, immunodeficiencies, and reproductive
immunology.206 Work in the horse continues in many of Proinflammatory Cytokines,

these areas from the standpoint of equine medicine and Acute Phase Proteins,
comparative immunology. With some exceptions, the over- and Complement
all organization and function of the equine immune sys-
tem is similar to other mammalian species. A number of After physical barriers have been breached, the innate immune
texts provide excellent in-depth reviews of basic and clini- system provides a variety of internal defenses to contain and
cal immunology, and much of the general information pro- eliminate the invading organisms. Inflammatory responses are
vided in this chapter is drawn from these resources.207-210 initiated either via the activation of plasma protease systems
This chapter will focus on aspects of the immune sys- directly, such as by bacterial cell wall components, or by the
tem that may be of most interest to equine clinicians and secretion of toxins or other proteins that can directly activate
researchers, with pertinent references to equine work pro- the inflammatory response.217 The cell walls and membranes
vided when possible. of bacteria contain various proteins and polysaccharides with
characteristic, often repeating, molecular structures. These
pathogen-associated molecular patterns (PAMPs) include
Innate Immunity and the Acute lipopolysaccharides (LPSs), peptidoglycans, lipoteichoic acid,
Inflammatory Response and flagellins.218 Other PAMPs include viral nucleic acids
Immune defenses include both innate responses and adap- and unmethylated bacterial cytosine-guanosine dinucleotides
tive responses, each mediated by cellular and soluble com- (CpG). PAMPs are recognized by pattern recognition receptors
ponents. Although fundamentally different, the innate and found on cells of the immune system, particularly those cells
adaptive immune systems are intimately related and share involved in the initial encounter with invading microbes. These
many of the same processes and components. Innate immu- “sentinel cells” include macrophages, dendritic cells, and mast
nity is rapid, is nonspecific, results in acute inflammation, cells, with macrophages being the most important for initiating
and has no memory. In contrast, adaptive immunity takes the inflammatory response. Pattern recognition receptors are
time to develop, is antigen specific, and has recall capability. located on the cell surface, as well as intracellularly, and include
It is both the specificity of adaptive responses and the capac- toll-like receptors (TLRs), retinoic acid–inducible gene–1
ity for immunologic memory that allow complete protection (RIG-1)–like receptors, and nucleotide-binding oligomeriza-
against a particular pathogen. However, innate responses tion domain–like receptors. The TLRs are particularly impor-
play key roles in both triggering the adaptive response and tant in the induction of inflammation. In addition, binding of
providing valuable time for specific adaptive responses to PAMPs leads to intracellular signaling events culminating in
develop. the expression of costimulatory signals for the developing adap-
The horse, like every other species, is under constant tive immune response. Injured cells release products collec-
assault from a variety of microbes that share its living space. tively termed damage-associated molecular patterns (DAMPs)
Although most of these organisms are thought to be harmless, that bind TLRs on macrophages and other cells leading to the
their disease-causing potential is evident when they cause production of proinflammatory cytokines that augment the
opportunistic infections in individuals with compromised inflammatory process. Resident macrophages that encounter
immune systems. In general, mammals have evolved a variety the invader initiate the inflammatory response through the
of defensive measures to prevent infections from occurring. production of proinflammatory cytokines such as interleu-
The first line of defense includes the physical barriers pro- kin-1 (IL-1), IL-6, and tumor necrosis factor–alpha (TNF-α).217
vided by the skin and the mucosal surfaces of the digestive, Cytokines are hormone-like proteins that mediate a variety of
respiratory, and urogenital tracts. In addition to providing a cellular responses. A vast number of cytokines are involved in
barrier to penetration, the surface of the skin contains vari- the regulation of innate and adaptive immune responses. IL-1,
ous enzymes, fatty acids, and oils that inhibit the growth of for example, is a pleiotropic mediator of the host response to
bacteria, fungi, and viruses. Mucous membranes and mucosal infections and tissue injury (Table 1.3). Many of the effects of
secretions contain bacteriolytic enzymes, bactericidal basic IL-1 are mediated through its capacity to increase the produc-
polypeptides, mucopolysaccharides, and antibodies that pre- tion of other cytokines, such as granulocyte colony-stimulating
vent colonization and penetration of these surfaces. Mucus factor (G-CSF), TNF-α, IL-6, and platelet-derived growth fac-
also provides a physical barrier that entraps invading organ- tor (PDGF). IL-6 is responsible for the increased production
isms and leads to their eventual disposal.211 Particles trapped of acute phase proteins by hepatocytes, including complement
in the mucus secretions of the respiratory tract, for example, proteins, C-reactive protein, and serum amyloid A (SAA).
are transported upward through the action of ciliary cells to
the trachea where they are swallowed.212 Once swallowed, TABLE 1.3 Biologic Activities of Interleukin-1
the acidic secretions and digestive enzymes of the stomach
destroy most organisms. Normal epithelial and tissue archi- Activates T cells Induces fever
tecture is essential for successful exclusion of bacteria, and Activates B cells Cytotoxic for some tumor cells
the disruption of this mechanism makes the host susceptible Enhances NK cell killing Cytostatic for other tumor cells
to infection by bacteria that normally colonize the upper Fibroblast growth factor Stimulates collagen production
airway.213,214 The protective role of gastric acidity against
Stimulates PGE synthesis Stimulates keratinocyte growth
gastrointestinal pathogens is supported by the observation
that acid-suppressive drugs increase the risk of diarrhea in Stimulates bone resorption Stimulates mesangial cell
neonatal foals treated in intensive care units.215 In humans, growth
administration of proton pump inhibitors in critically ill Chemotactic for neutrophils Activates neutrophils
patients increases the risk of Clostridium difficile–associated Activates osteoclasts Induces IL-6 production
diarrhea.216
Many of these proteins and the cytokines that elicited them are bound to the surface of a microbe. Bound C1q is proteolytic
responsible for the characteristic physical signs of inflamma- and cleaves other complement components activating the
tion, including increased blood flow and vascular permeability, pathway. The activation of complement via the alternate path-
migration of leukocytes from the peripheral blood into the tis- way does not involve antibodies. Instead, certain microbial
sues, accumulation of leukocytes at the inflammatory focus, and products stimulate the association of factor D, a proteolytic
activation of leukocytes to destroy any invading organisms.219 enzyme, with the complex of factor B and C3b leading to the
During viral infections, intracellular pattern recogni- formation of C3 convertase and its deposition on the micro-
tion receptors (such as RIG-1) bind to viral PAMPs (such as bial surface. All three pathways converge with the generation
double-stranded viral ribonucleic acid [RNA]) and induce the of C3 convertase and the cleavage of C3. Soluble C3a, pro-
production of type I interferons (IFNs), including IFN-α and duced by the cleavage of C3 by the C3 convertases, can bind
IFN-β. These are produced in virus-infected cells within hours to mast cells causing them to degranulate and is thus referred
and inhibit viral replication through various mechanisms. In to as an anaphylatoxin, as is C4a. C3b serves as an opsonin for
addition, natural killer (NK) cells are capable of lysing virus- C3b receptor-bearing phagocytic cells. C3b is also required for
infected cells via recognition of different types of receptors the formation of the membrane attack complex by the termi-
on the surface of infected cells, as well as recognizing cells nal complement components, C5 through C9. In this process
expressing fewer major histocompatibility complex molecules C5 is cleaved and C5a is generated, which, along with C3a, is a
(see later discussion), the expression of which can be down- chemoattractive factor for neutrophils and monocytes.221 C5b
regulated by viral infection. Finally, NK cells have surface Fc forms a complex with C6, C7, and C8 on cell surfaces. This
receptors (see later discussion) that can bind the Fc portion leads to the insertion and polymerization of C9 that forms a
of antibodies bound to the surface of infected cells and subse- pore in the membrane leading to cell lysis. A summary of the
quently kill the infected cell through the process of antibody- three complement pathways and the resultant effector mecha-
dependent cellular cytotoxicity. NK cells are of the lymphocyte nisms of microbial destruction is provided in Fig. 1.6.
lineage but do not possess antigen-specific receptors such as Lipid Mediators. Prostanoids are lipid mediators that regu-
T and B lymphocytes (discussed later) and thus act as innate late the inflammatory response.222,223 The prostanoids include
effector cells. the prostaglandins (PGs), leukotrienes (LTs), and prostacyclin
The complement system consists of over 30 different (PGI2), and they are the product of cyclooxygenase cleavage
plasma proteins that are produced primarily in the liver. This of arachidonic acid followed by endoperoxidation. The major
complex interacting series of proteases and their substrates sources of prostanoids in acute inflammation are the phago-
results in the production of physiologically active interme- cytes, endothelial cells, and platelets. Although prostanoids, in
diaries that can damage membranes, attract neutrophils and general, mediate the cardinal effects of pain, fever, and edema
other cells, increase blood flow and vascular permeability, and characteristic of the acute inflammatory response, their par-
opsonize bacteria and other particles for phagocytosis.220 The ticular roles are somewhat confounding and can be either pro-
complement cascade can be activated in three ways. The lectin inflammatory or antiinflammatory.224 Prostanoid production
pathway is initiated when soluble carbohydrate-binding pro- depends on the activity of the two isoforms of the cyclooxy-
teins (such as mannose-binding lectin) bind to carbohydrate genase (COX) enzymes within cells: COX-1, which is present
structures on microbial surfaces. Proteases associated with in most cells and its expression is generally constitutive, and
these carbohydrate binding lectins then initiate the cleavage of COX-2, for which expression is low or undetectable in most
complement components and activate the pathway. The classi- cells but its expression increases dramatically on stimulation,
cal pathway involves the binding of C1q to antibodies already particularly in cells of the immune system. Increased COX-
Lectin Pathway Classical Pathway Alternate Pathway

Lectins bind carbohydrates C1q binds antibody bound C3 convertase deposited
on microbial surface to microbial surface directly on microbial surface
Generation of C3 Convertase
C3 is cleaved, leaving C3b bound
to microbial surfaces and
releasing soluble C3a
Chemotaxis Opsonization Microbial Lysis

Soluble C3a and C5a recruit Phagocytic cells with C3b Assembly of components C5-C9
phagocytic cells to site of receptors engulf and destroy on the microbial surface leads to
infection and promote microbes with surface-bound formation of the membrane attack
inflammation C3b complex and lysis of the microbe
FIG. 1.6 Simplified pathways of complement activation and basic effector mechanisms of microbial de-
struction. See text for explanation.
2 expression by inflammatory stimuli likely accounts for the ous chemotactic factors by host cells, bacteria, and other in-
high levels of prostanoids found in inflammatory lesions and vaders causes various leukocytes to enter the circulation and
is the basis for the development of COX-2–selective nonsteroi- be carried to the site of the injury.238 Chemokines are chemo-
dal antiinflammatory drugs (NSAIDs).225 Because there are tactic cytokines. These are soluble proteins produced by host
species differences in the pharmacokinetics and COX selec- cells that induce the directional migration and activation of
tivity of NSAIDs, extrapolation of studies performed in other leukocytes, as well as other somatic cell types, and thus play
species should be interpreted with caution with respect to the major roles in the inflammatory response.239 The chemokine
potential COX selectivity and efficacy in the horse.226 Meloxi- CXCL8 (previously known as IL-8) plays a central role in the
cam has COX-2 selectivity and efficacy in the horse227-229 but migration of neutrophils, including equine neutrophils.240,241
at the time of this writing is not approved in the United States Other chemokines promote humoral and cell-mediated im-
for use in horses (but is approved in Europe). Firocoxib is a mune reactions; regulate cell adhesion, angiogenesis, leuko-
selective equine COX-2 inhibitor with documented safety and cyte trafficking, and homing; and contribute to lymphopoiesis
efficacy,210-236 and it is currently the only COX-2–selective in- and hematopoiesis.242
hibitor approved by the Food and Drug Administration for The specific trafficking of leukocytes from the blood to
use in horses in the United States. inflammatory sites is dependent on both the production of
Both COX isoforms produce PGH2, which is the common chemotactic factors and the interaction of specific receptors
substrate for a series of specific synthase enzymes that pro- on the leukocytes with corresponding adhesion molecules on
duce PGD2, PGE2, PGF2, PGI2, and TXA2. It is the differen- the endothelial surface of the blood vessels. Neutrophil adher-
tial expression of these enzymes within cells present at sites of ence is a two-step process first involving endothelial cell sur-
inflammation that determines the profile of prostanoid pro- face molecules known as selectins. Small venular endothelium
duction. Likewise, the biologic effect of a prostanoid depends overlying a site of inflammation and exposed to leukotrienes,
on binding to G protein–coupled cell surface receptors. platelet activating factor (PAF), IL-1, complement protein
Many cells of the immune system express multiple receptors C5a, histamine, LPSs, TNF-α, or other mediators released
that couple to apparently opposing pathways. The impact of by clotting, platelet activation, or mast-cell activation express
prostanoids present during an inflammatory response is thus P-selectin. P-selectin mediates the process in which neutro-
determined by the array of receptors the cells express and the phils initially interact with the endothelial surface in a pro-
intracellular pathways to which they are coupled. Activation cess known as “rolling” in which the circulating neutrophil
of these receptors, even when coupled to similar pathways, interacts with the endothelial cell before the actual adher-
might evoke different responses because of differences in the ence. P-selectin appears on the endothelial cell surface within
levels of expression (both constitutive and induced) or in the minutes of an inflammatory stimulus, followed by E-selectin
patterns of desensitization. The role of prostanoids in a given expression within a few hours. Endothelial selectins function
inflammatory response depends not only on the presence of by binding to carbohydrate ligands present on the leukocyte
the lipid mediators in the lesion but also the receptor profile surface. In the case of neutrophils, the ligand is sialyl-Lew-
on immune cells and the biochemical signaling pathways of isX, an oligosaccharide present on cell surface glycoproteins.
these receptors.237 Thus PGE2 is considered proinflammatory Equine P-selectin binds the sialyl-LewisX moiety on equine
because it promotes vasodilation by activating receptors on leukocytes, mediating adhesion and stimulating the produc-
vascular smooth muscle and increases vascular permeability tion of CXCL8.243 The second part of the adherence process
indirectly by enhancing the release of histamine and other is the tight binding of integrins on the neutrophil surface to
mediators from tissue leukocytes such as mast cells. PGE2 is intracellular adhesion molecules on the endothelial cell sur-
also the prostanoid responsible for fever production. However, face. Leukocyte integrins are heterodimeric proteins and
as inflammation progresses, PGE2 synthesis by macrophages include leukocyte functional antigen–1 (LFA-1, also called
is enhanced as a result of increased expression of COX-2 and CD11a:Cd18) and complement receptor type 3 (CR3, also
PGE-synthase, and the resulting increased levels of PGE2 called CD11b:Cd18). Neutrophils can be activated by a num-
inhibit leukocyte activation, inhibit mast cell degranulation, ber of soluble proteins present in bacterial but not eukaryotic
and relax smooth muscle contractions. In the lung, PGE2 pro- proteins. Host factors present at the site of inflammation can
motes bronchodilation. Thus in these situations PGE2 may be also activate neutrophils. These include complement proteins
considered antiinflammatory. (C5a, C3a), the chemokine CXCL8, cytokines (especially TNF-
Chemotaxis and Leukocyte Trafficking. One of the initial α), and immune complexes.244 Expression of integrins by the
and most crucial aspects of the acute inflammatory response activated neutrophil allows them to become tethered to the
is the recruitment of leukocytes (primarily neutrophils) to the endothelial surface. The migration of neutrophils through the
site of injury. Neutrophils constitute the first line of the cel- vascular wall is less well understood than these initial events
lular defense and are the initial cells involved in an inflam- leading to firm adhesion. Integrins LFA-1 and CR3, as well as
matory response. These phagocytic cells are derived from PECAM-1 (CD31), another cell adhesion molecule, appear to
multipotent stem cells located chiefly in the bone marrow. play a role in this process. Endothelial cell–produced CXCL8
Under the influence of a variety of signals provided from both also has a critical role in this process. Once through the endo-
within and outside of the bone marrow, these stem cells be- thelium, the phagocytes will follow chemotactic signals and
come committed to developing into cells of the granulocyte migrate toward the point of injury. They may adhere to other
lineage. The critical signal is provided by a family of growth cells during migration to the site of inflammation, and these
factors known as colony-stimulating factors (CSFs) that pro- interactions also are dependent on integrins.
vide both proliferative and differentiating signals leading to Neutrophils recruited and activated in this manner will
the development of granulocytes and other leukocytes. Once actively phagocytose microscopic invaders and attempt
released into the circulation these cells must find their way to to destroy them using reactive oxygen products gener-
the site of the inflammatory response. The production of vari- ated via a nicotinamide adenine dinucleotide phosphate
(NADPH)-oxidase–dependent respiratory burst. During Adaptive Immunity

this process, neutrophils release additional proinflammatory The adaptive immune response is initiated in response to
mediators, thus amplifying the response. Among those cells the encounter with a foreign agent and depends on antigen-
attracted to the area are NK cells, and the production of IFN- specific immune responses mediated by different divisions
α or IFN-β by macrophages and other cells enhances NK cell of the lymphocyte family (Fig. 1.7). In contrast to the non-
cytolytic activity. The NK cells themselves can be a source specific nature of the innate immune response, an important
of IFN-γ, another proinflammatory cytokine (discussed in characteristic of the adaptive immune response is the speci-
detail later). Depending on the magnitude of the initial insult ficity of this interaction. Thus exposure of the host to a par-
and the susceptibility of the invader to neutrophil-mediated ticular microbe or parasite results in the induction of immune
destruction, the inflammatory response may be either acute responses that are directed against specific components of the
or chronic. invading organism that do not affect unrelated organisms. The
Acute inflammation is thus a rapid response to an injury that specificity of the adaptive immune response is the result of the
is characterized by accumulations of fluid, plasma proteins, and interaction of specific molecular structures, or antigens, of the
neutrophils that rapidly resolves once the initial inflammatory invader with antigen-specific receptors on lymphocytes. All
stimulus is removed. Deactivation signals include PGE2, corti- types of chemical structures can serve as antigens, including
sol, IL-10, and transforming growth factor–β (TGF-β). Some of proteins, nucleic acids, lipids, and polysaccharides. However,
those chemotactic agents responsible for initiating the response foreign molecules that are large, complex, and stable are the
(CXCL8, FMLP, C5a, LTB4, and PAF) also serve to downregu- most antigenic. Large antigens, such as proteins, contain mul-
late its intensity by inducing the shedding of IL-1 receptors tiple antigenic determinants or epitopes that interact with lym-
from neutrophils.245 The shedding of this decoy receptor may phocytes via their antigen-specific receptors. Haptens consist
have antiinflammatory effects as it effectively binds and neutral- of single antigenic determinants and can effectively combine
izes this cytokine. Likewise many of the acute phase proteins with the binding site of antibody molecules. However, as they
are thought to have immunomodulatory activity downregulat- only consist of a single antigenic determinant, they cannot
ing neutrophil function.246 Acute inflammatory responses may cross-link B-cell receptors (antibody molecules), and they are
often be subclinical and resolve without complications. How- also unable to stimulate T-cell responses. Therefore haptens
ever, if the invader is resistant to neutrophil-mediated destruc- cannot stimulate an immune response unless multiple hap-
tion or the degree of injury is large, the response may become tens are physically attached to a larger molecule, known as a
more chronic with the added recruitment of macrophages, lym- carrier.
phocytes, and fibroblast growth. Like the innate response, the adaptive immune response to
The essential characteristic of the innate immune response a specific antigen consists of both humoral and cellular effector
is that it does not exhibit specificity for the invading organ- mechanisms. The humoral component is mediated by immu-
ism. Thus the induction of an innate immune response does noglobulins or antibodies found in plasma and tissue fluids.
not require prior exposure to the invading organism nor is Antibodies are produced by B lymphocytes, small lymphoid
it augmented by repeated exposure to the same organism. In cells characterized by the cell surface expression of immuno-
most instances these mechanisms are adequate for eliminating globulin molecules. B cells represent less than 15% of the cir-
casual invaders. However, pathogenic organisms have evolved culating peripheral blood mononuclear cells but are present
various methods for avoiding elimination. In response to these in higher proportions in lymph nodes and the spleen. B cells
organisms, the specialized cells and products of the adaptive are derived from the fetal liver and bone marrow of mammals
immune response are mobilized. and the bursa of Fabricius of birds. In the bone marrow, B
CD4 TH1 IFN-γ

Helper
TCR T cell
CD3 IL- 4, IL- 5
TH 2
IL -13, IL-10
T cell
CD8
TH17 IL-17, IL- 22
Lymphoid Cytotoxic
progenitor T cell
Treg TGF- β , IL-10
Plasma YY
sIgM sIgG /A/E cell Y
YY
B cell
Memory
B cell
Distinguished by cell surface molecules Functional distinctions
FIG. 1.7 Major divisions of the lymphocyte family. To the left of the diagram different populations of lymphocytes are distinguished by expression of
different cell surface molecules. To the right of the diagram the distinctions are functional.
Light chain
VL
CL Secretory piece
VH C 2
C 3
C 1
Heavy chain
J chain
Variable region Constant regions Fc region Fab region

(antigen (effector
recognition) functions)
FIG. 1.8 Molecular structure of secretory IgA. This schematic illustrates the major features of immunoglobu-
lin molecules. Whereas the illustrated IgA molecule is dimeric, with the two Ig units joined by a “J chain” and
a series of disulphide bonds, IgG molecules are monomeric. Each Ig unit consists of two heavy chains and
two light chains. The heavy chains have four subunits and the light chains two. One end of the Ig unit has a
highly variable protein structure and is involved in antigen recognition, and the remainder of the Ig unit has a
constant structure in each Ig class and subclass and determines the functional characteristics of the molecule,
such as binding complement, or recognition by macrophages or neutrophil Fc receptors. This specialized
dimeric IgA molecule also has a secretory piece that increases its stability in the harsh mucosal environment.
In this diagram, the Fc portion is depicted as a circular structure, consistent with actual x-ray crystal structure
determinations.
cells are the products of a putative lymphoid stem cell derived infection and/or disease (the basis of vaccination). Although
from the pluripotent stem cell. Under the influence of various this principle appears to be straightforward, vaccination
cytokines produced by bone marrow stromal cells, the B-cell does not always yield the expected result. Why some vac-
precursor undergoes its 3-day development into a mature B cines work and others fail is a complex issue, a major compo-
cell. On stimulation with a specific antigen, B cells differentiate nent of which is the nature of the antigen-specific receptors
into plasma cells that produce enormous quantities of a spe- of lymphocytes.
cific antibody. The activation, proliferation, and differentiation
of B lymphocytes into plasma cells is dependent on other cells, Immunoglobulin: Antigen-Specific Receptor
including T lymphocytes, which represent the cellular com- of B Lymphocytes
ponent of the adaptive immune response. The T lymphocyte The antigen-specific receptor of the B cell is cell surface bound
is also derived from the multipotent stem cell and lymphoid immunoglobulin, also known as antibody. An immunoglobu-
precursor in the bone marrow, though its subsequent devel- lin molecule is composed of two identical light chains and two
opment into the mature T cell occurs in the thymus. Within identical heavy chains that form a disulfide-linked Y-shaped
the thymic environment the prothymocyte undergoes a devel- molecule (Fig. 1.8). The light chain can be divided into two
opmental and selective process while emigrating through the domains, a conserved carboxy-terminal domain and a highly
cortex into the medullary region of the thymus. Less than 3% variable amino-terminal domain. Analysis of heavy chains
of all the immature thymocytes found in the cortex survive to reveals a similar domain structure with the amino-terminal
become peripheral T cells. domain being highly variable and the presence of three con-
Though the induction of an antibody response requires stant domains. The antigen-binding region of an immuno-
the interaction of B and T lymphocytes, these cells recognize globulin molecule is formed by the association of the amino
different epitopes on the same antigen. Indeed, antigen rec- ends of a light and a heavy chain, and the carboxyl end of the
ognition by B cells and T cells is fundamentally quite differ- heavy chain determines the isotype of the molecule. Similar to
ent. B cells, and antibodies, recognize antigens in solution or most species, five different immunoglobulin isotypes (classes)
on cell surfaces in their native conformation, whereas T cells have been identified in the horse: IgM, IgD, IgG, IgE, and IgA
only recognize antigen in association with self molecules (Table 1.4).247-250 Before the availability of genetic character-
known as major histocompatibility complex molecules found ization of the equine immunoglobulin heavy chain gene loci,
on most cells surfaces (see later discussion). The adaptive at least four IgG subclasses were identified by physicochemical
immune response thus differs from innate immunity in that means and defined serologically by monoclonal antibodies as
it is antigen driven, and those cells that mediate the adap- IgGa, IgGb, IgGc, and IgG(T).251 More recently, seven IgG sub-
tive immune responses, T and B lymphocytes, express spe- classes have been defined (IgG1–IgG7) and named based on
cific receptors for the antigen. Because the immune system the designation of the corresponding Ig heavy chain constant
will respond to the antigens of both live and killed patho- region genes.248,250 The original IgGa corresponds to IgG1,
gens, it is possible to stimulate immunity without causing IgGb to both IgG4 and IgG7, IgGc to IgG6, and IgG(T) to both
TABLE 1.4 Immunoglobulin Isotypes

Isotype Immunologic Function
IgM Surface IgM is found on naive, activated, and memory B cells. Secreted IgM is a pentamer and represents the major
antibody produced during a primary response. IgM efficiently mediates agglutination, neutralization, opsonization, and
complement activation.
IgD Antigen receptor of naive B lymphocytes. An IgD heavy chain gene has been identified in the horse, and it is expressed.
IgG The principal immunoglobulin found in plasma representing up to 80% of the total immunoglobulin concentration.
Seven subclasses of IgG have been identified (see text). The major functions of IgG include opsonization and neutral-
ization reactions. IgG1, IgG3, and IgG4/7 are effective in fixing complement, and IgG1, IgG4/7, and IgG3/5 bind Fc
receptors so they can function as opsonins and mediate antibody-dependent cellular cytotoxicity. IgG6 and IgG5 may
play an important role in exotoxin neutralization and immunity to parasites.
IgE Most IgE is found associated with the surface of mast cells and basophils and only very small amounts are present in
the plasma. The cross-linking of two IgE molecules with specific antigen results in the degranulation of the mast cells
and basophils. Thus IgE is the primary antibody responsible for type I hypersensitivity reactions and appears to play a
central role in immunity to parasites.
IgA The most abundant antibody in secretions (tears, mucus, saliva, colostrum, etc.) is a dimer composed of two IgA mol-
ecules joined by a J chain. IgA in the plasma is predominantly monomeric. IgA antibodies can be neutralizing but do
not fix complement efficiently nor do they have efficient opsonization activity.

IgG3 and IgG5.247,250 All seven IgG subclasses are expressed within the lymph nodes and spleen (and other secondary
and functional, with IgG1, IgG3, IgG4, IgG5, and IgG7 able to lymphoid organs) during a primary immune response and
elicit a respiratory burst in equine peripheral blood leukocytes requires interaction with T lymphocytes. Cytokines secreted
(predictive of binding to Fc receptors), whereas IgG1, IgG3, by these interacting helper T cells provide the signals driv-
IgG4, and IgG7 bind complement C1q and activate the clas- ing class switching. For example, IL-4 and IL-13 induce
sical complement pathway.247 Some functional discrepancies isotype switching to IgE, and interferon-gamma (IFN-γ)
exist between the new and original designations. For example, inhibits this induction and augments IgG production. IgA
IgG(T) does not fix complement and inhibits complement is produced in response to the combination of the cytokines
fixation by IgGa (IgG1) and IgGb (IgG4/7).252 This is not con- IL-4, IL-5, and transforming growth factor-β (TGF-β).
sistent with the complement fixing properties of IgG3, which, The antigen specificity of a particular antibody molecule
along with IgG5, corresponds to IgG(T).247 Perhaps new (and the B cell that produces it) is determined by the combi-
reagents will provide clarification. nation of the variable domains of the light and heavy chains.
Membrane-bound immunoglobulin serves as the anti- The association of these two domains results in the formation
gen-specific receptor for B lymphocytes (B-cell receptor of an antigen-binding cleft or pocket that contains regions of
[BCR]). Thus antibodies in serum are secreted soluble forms hypervariability that define the specificity of a particular anti-
of BCRs. Each BCR contains a membrane-spanning region body molecule. It has been estimated that over 108 different
near its carboxy end that is inserted into the mRNA dur- antibody specificities are possible. The generation of this tre-
ing differential splicing of the heavy chain exons. Membrane mendous amount of diversity in antibody specificity occurs
IgM and IgD comprise the BCRs on the surface of naive B during B-cell ontogeny in the bone marrow. Within a given
lymphocytes, although the function of IgD is unclear. The B cell, the genes encoding the heavy and light chains of an
IgD isotype is secreted in very small quantities, if at all, and antibody molecule are organized into specific gene segments.
is rarely detectable in the circulation. Once activated, B cells Thus the light chain is formed from variable (VL), joining (JL),
cease to express IgD but continue to express the membrane and constant (CL) gene segments, which together form the
form of IgM. Early on in an immune response the B cell variable and constant domains of the light chain. In the germ
secretes large amounts of the pentameric form of IgM. As the line of an undifferentiated human cell one finds several hun-
immune response proceeds, the B cell will switch the isotype dred different VL and several dozen JL gene segments. In the
of its heavy chain. Isotype switching involves the substitu- horse, there are 204 VL genes, 12 JL genes, and 8 CL genes.253
tion of one heavy chain constant region in place of another. Likewise the heavy chain of a B lymphocyte is composed of
In the horse, the genes encoding the 11 different constant VH, diversity (D), and JH segments, which form the variable
(C) regions (five primary isotypes including the seven IgG domain, and these join to the constant region genes (discussed
subclasses) of the heavy chain are sequentially arranged earlier) to form the complete heavy chain molecule. Similarly,
on chromosome 24 in the following order: Cμ(M), Cδ(D), in the germ line of humans one finds a large number of VH
Cγ1(G1), Cγ2(G2), Cγ3(G3), Cγ7(G7), Cγ4(G4), Cγ6(G6), gene segments and a smaller number of DH and JH segments.
Cγ5(G5), Cε(E), Cα(A).250 Initially, the first two constant There are 54 VH, 40 DH, and 8 JH genes in the horse.253 During
region genes encoding the M and D constant regions are the differentiation of a B cell (Fig. 1.9), there is the sequential
used to form the heavy chain. When switching occurs a new selection and rearrangement of a VL segment with a JL seg-
constant region segment is selected, and the intervening ment and the accompanying deletion of intervening VL and
genes are removed either by splicing or looping out. Isotype JL segments (Fig. 1.10). The VJ segment is joined with a C
switching only affects the heavy chain constant domains and gene, and the rearranged VJC sequence is then transcribed
has no effect on the antigen specificity of the immunoglobu- into mRNA and translated into the light chain. A somewhat
lin molecule. Isotype switching occurs in lymphoid follicles similar sequence follows for heavy chains except that two
Plasma
Lymphoid Pro-B Pre-B Immature Mature Activated cell
stem cell cell cell B cell B cell B cell
Ig gene rearrangement Antigen driven
cytoplasmic µ surface surface surface

IgM
(IgM heavy chain) IgM & D IgG/A/E
Memory
B cell
FIG. 1.9 B-cell differentiation. Different stages of B-lymphocyte development can be recognized by expression of immunoglobulin molecules. This
maturation requires a series of gene rearrangements to select the genes that will encode the antigen binding part of the immunoglobulin molecule (vari-
able region), and subsequently to select the genes that determine the class or subclass of the antibody molecule. Initially immature B cells express IgM
(the majority of peripheral blood B cells), but after antigen exposure the B cell becomes activated and may express any of the immunoglobulin classes or
subclasses. This decision depends in large part on cytokine signals from helper T cells. Activated B cells either mature into short-lived antibody-secreting
plasma cells or become long-lived memory B cells.
Variable region genes Constant region genes
IgGa ? IgG(T) IgGb IgGc IgG(T)

IgM IgD IgE IgA
V1 2 3 4 5 D1 2 3 J1 2 Cµ Cδ Cγ1 Cγ2 Cγ3 Cγ7 Cγ4 Cγ6 Cγ5 Cε Cα
Germline DNA
V1 2 3 4 5 D1 J2 Cµ Cδ Cγ1 Cγ2 Cγ3 Cγ4 Cγ5 Cγ6 Cγ6 Cε Cα

DJ joined rearranged DNA
V5 D1 J2 Cµ Cδ Cγ1 Cγ2 Cγ3 Cγ4 Cγ5 Cγ6 Cγ6 Cε Cα

VDJ rearranged DNA
V5 D1 J 2 Cγ4
VDJ, constant region rearranged DNA
Complete IgGb heavy chain polypeptide chain
Variable region Constant regions
FIG. 1.10 Immunoglobulin gene rearrangement: somatic recombination process for production of an
immunoglobulin heavy chain. The figure shows a hypothetical series of V, D, and J variable heavy chain
genes, positioned 5′ to the known equine heavy chain constant region gene loci. In the first step in somatic
recombination a D and a J gene segment are joined, and in the second step a V gene segment is joined to
complete the VDJ recombination and form a gene capable of encoding the variable region. Subsequently
one of the seven equine γ heavy chain constant regions, labeled with their corresponding IgG subclass,
was selected to complete the gene rearrangement. Because the Cγ4 heavy chain constant region gene was
selected, this leads to production of an IgG4 heavy chain.
rearrangements are necessary, a D to J rearrangement fol- of RAG-1 and RAG-2 components, encoded by recombina-
lowed by a V to DJ rearrangement. Once completed, the VDJ tion-activating gene–1 and recombination-activating gene–2.
segment is brought into proximity of the appropriate CH seg- To reconstitute the rearranged gene the cut ends of the DNA
ment and transcribed. During gene rearrangement, interven- must be rejoined, which requires the enzyme DNA-depen-
ing deoxyribonucleic acid (DNA) is looped out and excised, dent protein kinase (DNA-PK). Not all of the gene segment
which requires V(D)J recombinase. This enzyme is made up rearrangements produce functional genes. Because a B cell
has two sets of heavy chain genes, one on each chromosome, Analysis of the predicted amino acid sequences for the
and most species have two different sets of light chain genes, TCR proteins confirmed a structural similarity with antibody
including the horse,253-255 there are several chances to form molecules. One peculiarity in the structure of the TCR was
appropriate heavy and light chains. Once the heavy and light observed from the amino acid sequence analysis. Although
chain gene segments are successfully recombined, the genes both the α and β chains of the TCR contained a transmem-
on the sister chromosome neither recombine nor are they brane region, both proteins had very short cytoplasmic tails.
expressed. This process of allelic exclusion ensures that the However, the TCR heterodimer is noncovalently associated
B cell produces antibodies of a single specificity. Although with the CD3 complex of proteins. The five proteins of the
this random assortment of gene segments accounts for much CD3 complex are involved in signal transduction following
of the diversity in antibody specificity, additional mecha- TCR binding to antigen. Unlike the TCR α and β proteins,
nisms are also involved, including gene conversion (inser- the CD3 proteins have large intracellular domains, some of
tion of pseudogenes), junctional diversity (resulting from the which are phosphorylated in response to stimulation of the
imprecise joining of gene segments), and somatic mutations. TCR (see later discussion). In addition to providing a sig-
Somatic mutations are point mutations in the hypervariable naling mechanism for the TCR, the CD3 complex is also
region of either the heavy or light chain that occur during the required for the expression of the TCR heterodimer on the
proliferation of antigen-activated B lymphocytes within ger- cell surface.257
minal centers of lymph nodes and other secondary lymphoid The generation of diversity in the TCR during T-cell
organs and tissues. Such mutations play an important role in ontogeny employs a mechanism similar to that used to gen-
increasing antibody affinity for its antigen. Thus fewer than erate immunoglobulin diversity. The TCR α chains resemble
500 genes can give rise to over 108 molecules of the various immunoglobulin light chains in that they are composed of
specificities needed to recognize the vast number of antigens V, J, and C gene segments. The particular V, J, and C seg-
the host may encounter. ments used are selected from a germ line configuration con-
taining a few (C region) to several hundred (V region) gene
T-Cell Receptor and CD3 Complex: Antigen- segments. The selection and rearrangement of the gene seg-
Specific Receptor of T Cells ments are similar to those employed by the immunoglobu-
T lymphocytes can be differentiated from B lymphocytes lin light chain and appear to involve the same recombinases
in that they do not express surface immunoglobulins but and DNA-PK. Likewise the β chains resemble heavy chains,
instead express the T-cell receptor (TCR). T cells also each being composed of V, D, J, and C gene segments, and
express another surface molecule called CD3. (The desig- their selection and rearrangement from germ line genes also
nation CD stands for cluster of differentiation and is the parallels immunoglobulin heavy chain rearrangement. Thus
result of an international workshop to standardize the ter- the generation of diversity is the result of the combination
minology used to describe leukocyte surface antigens rec- of multiple gene segments and junctional diversity. However,
ognized by monoclonal antibodies.) The TCR and CD3 unlike immunoglobulins, the TCR genes do not undergo
form a multimeric complex on the T-cell surface, and this gene conversion or somatic mutations.
complex is involved in antigen-specific recognition. The
TCR was originally described as a heterodimer comprised T-Lymphocyte Subsets
of an α chain and a β chain. Peptide mapping studies of Mature T lymphocytes can be further divided into two dis-
the α and β chains from many different T-cell lines demon- tinct populations on the basis of their expression of either
strated that they contained variable and constant domains the CD4 or CD8 molecule.258 The expression of these surface
reminiscent of immunoglobulin structure. Further analy- molecules is directly correlated with the specificity of the T
sis indicated that, like immunoglobulin genes, TCR genes cell (see later discussion). The expression of either CD4 or
undergo gene rearrangements during T-cell development to CD8 also correlates with T-cell function. Thus those cells that
generate tremendous diversity in antigen specificity. Sub- express CD8 are typically cytotoxic effector cells (cytotoxic T
sequently, two additional TCR genes were identified, the γ lymphocytes [CTLs]), whereas those that express CD4 are
chain and δ genes corresponding to a second heterodimer. typically helper cells that produce cytokines that enhance
Thus two types of TCRs exist, an αβ heterodimer and a γδ antibody and cell-mediated immune responses. Whereas the
heterodimer. Although the percentages of T cells expressing T lymphocytes in the periphery express either CD4 or CD8,
the γδ receptor vary among mammalian species (ruminants cortical thymocytes (immature T cells in the thymus) express
and pigs have higher percentages of γδ T cells than other both antigens. During maturation in the thymus, these cells
species), most T cells are αβ T cells with fewer than 5% of convert to either CD4+ or CD8+ cells or they are eliminated
all T cells expressing the γδ receptor. The function of γδ T (Fig. 1.11). Within the cortex of the thymus, thymocytes are
cells is poorly understood. In general, γδ T cells generate exposed to thymic epithelial cells that present self peptides
less diversity in their TCRs, can recognize nonpeptide anti- in the context of major histocompatibility complex (MHC)
gens such as lipids, do not require major histocompatibility molecules (see later discussion). The thymic selection pro-
complex presentation for antigen recognition (see later dis- cess involves both positive and negative selection. During
cussion), and participate in inflammatory responses. They positive selection, thymocytes with TCRs that bind self pep-
secrete cytokines and can have cytotoxic activity and may tide–MHC complexes with low avidity (weakly) are stimu-
be important for recognizing antigens frequently encoun- lated to survive. This ensures that the developing thymocyte
tered at mucosal surfaces and epithelial boundaries, at the has a functional TCR. During negative selection, thymocytes
interface between the host and the external environment.207 that bind self peptide–MHC complexes with high avidity
As such they are thought to play an important role in immu- (strongly) are deleted. The end result is that mature T lym-
nologic surveillance. In the horse, CD8+ T cells expressing phocytes leave the thymus with functional TCRs capable of
the γδ TCR have been identified in peripheral blood.256 binding self MHC molecules without autoreactivity.
{
Capsule
Immature CD4-/CD8- double negative

thymocytes proliferate and rearrange their
T-cell receptor genes
Cortex Immature CD4+/CD8+ thymocytes interact

with the network of thymic cortical epithelial
cells, which express MHC I & II molecules
{
Thymocytes with receptors with too high
an affinity for self-peptides or too low an
affinity for self-MHC molecules are
negatively selected and eliminated by
apoptosis
Medulla
CTL TH Mature single-positive (CD4 or CD8)
thymocytes reach the cortex and enter the
bloodstream as T cells
FIG. 1.11 T-cell maturation and selection in the thymus.
Major Histocompatibility Complex MHC I molecules are cell surface glycoproteins consisting
Molecules and Antigen Presentation of two noncovalently associated proteins, an MHC-encoded
Unlike B cells and antibodies, which recognize antigens in α chain (consisting of α1, α2, α3, transmembrane, and cyto-
solution or on cell surfaces in their native conformation, T plasmic domains) and β2-microglobulin, a protein encoded
cells only recognize processed antigen bound by antigen- outside of the MHC that stabilizes the molecule on the cell
presenting molecules on the surface of antigen-presenting surface (Fig. 1.12). The α1 and α2 domains are variable regions
cells (APCs). These antigen-presenting molecules are glyco- encoded by polymorphic genes and form the antigen bind-
proteins encoded by genes within a large gene cluster called ing cleft of the molecule. MHC I molecules are expressed on
the major histocompatibility complex (MHC), which also hap- the surface of most nucleated cells, with the highest level of
pens to be the most polymorphic gene region known in ver- expression on lymphoid cells and lower expression on fibro-
tebrates. The MHC was originally defined in terms of its role blasts, muscle cells, and neural cells. MHC I molecules are not
in allograft rejection. Following the rejection of a primary detectable on early embryonal cells, placental cells, and some
allograft, antibodies that reacted with the allograft could be carcinomas. The level of expression of MHC I molecules can
found in the recipient’s sera. These antibodies could be used to be modified by treatment with cytokines or infection with
identify or type tissues to determine the suitability of a donor viruses, including equine herpesvirus–1 (EHV-1), which
for transplantation. Genetic analysis of the MHC region downregulates expression.261 Interferons and TNF-α augment
demonstrated that there were a number of closely linked MHC I expression. This augmented expression is the result of
genes encoding several different, though related, molecules increased production of MHC I mRNA, and the regulatory
that were involved in allograft rejection. These closely related region of the MHC I genes has been shown to contain inter-
genes are collectively referred to as MHC class I (MHC I) feron and TNF-α–responsive elements that control the tran-
genes and their products as MHC I molecules. Similar to the scriptional activity of these genes.
anti–donor MHC antibodies found in allograft recipients, it The MHC I region of most species contains a large num-
was also demonstrated that multiparous mares had antibod- ber of polymorphic genes within several different loci (loca-
ies in their sera as a result of the exposure to paternal MHC tions on the chromosome). In humans there are three MHC
antigens on the fetus.259,260 These sera provided the earliest I loci (A, B, and C) with 2000 to 3000 expressed alleles iden-
means to serologically define MHC I haplotypes (the com- tified for each locus (Immuno Polymorphism Database;
plement of MHC alleles in a given individual) in horses.259 http://www.ebi.ac.uk/ipd). Comparably very few MHC I gene
Equine MHC I haplotypes are designated using the equine sequences are known in the horse. A recent BLAST query
leukocyte antigen (ELA) prefix, similar to the nomenclature (February 2016) returned approximately 100 equine MHC
used in humans and other species (i.e., “HLA” in humans). I sequences, and fewer have been published (approximately
In addition to the serologically defined MHC I molecules, 60).262-264 Although seven loci were identified in MHC I homo-
another group of molecules were identified within the MHC zygous horses (ELA-A3 haplotype),264 a more recent study in
that were involved in the stimulation of mixed lymphocyte horses representing 10 different haplotypes revealed that the
responses and the control of immune responsiveness. These number of loci differs by haplotype, with 4 confirmed and 3
MHC class II (MHC II) molecules are structurally and func- provisional loci identified.263 MHC I typing methods in horses
tionally distinct from the MHC I molecules, but both are include serologic typing,259 reverse transcriptase polymerase
involved in T-cell recognition of antigen. chain reaction (RT-PCR) cloning and sequencing to identify
Peptide
Antigen
α2 α1 β1 α1
α3 β2 α2
β2 -micro-
Trans- globulin
membrane
domain
Cell Surface
Cytoplasmic
domain
A B C
FIG. 1.12 MHC class I and II molecules . (A) Schematic depiction of an MHC molecule expressed on the cell
surface, with peptide bound in the cleft formed by the α1 and α2 domains. (B) Molecular model of equine
MHC class I molecule 7 to 6 (Eqca-N*00602) presenting the Rev-QW11 peptide, a known CTL epitope within
the Rev protein of equine infectious anemia virus. β2 microglobulin not shown. (C) Schematic diagram of an
MHC class II molecule expressed on the cell surface, with peptide bound in the cleft formed by the α1 and
β1 domains.
specific expressed alleles,263,265 and DNA microarray.63 Most Once in the ER, peptides are handed off to newly formed
recently, MHC haplotypes have been identified using micro- MHC I molecules and stabilize a trimolecular complex with
satellite markers.266,267 β2 microglobulin. This complex is then transported to the
MHC I polymorphism is primarily localized in the α1 and cell surface, where antigen presentation occurs. Because this
α2 domains, with the α3 domain being more conserved. The is a normal cellular process for eliminating degraded proteins
polymorphism of these two domains is related to their role in from the cell it is not surprising that MHC I molecules are nor-
presenting antigen to T cells. The physiologic role of MHC I mally loaded with these self-peptides. Indeed it is this encoun-
molecules was defined when it was discovered that CTL lysis ter with MHC I loaded with self-peptides in the thymus that
of virus-infected cells was restricted to target cells expressing is responsible for the deletion of autoreactive clones during
the same MHC I molecules as the CTL.268 This observation T-cell ontogeny. This unique peptide-binding characteristic of
led to the realization that T cells recognized the combination MHC I molecules has led to their use as immunologic reagents
of self-MHC and foreign antigen. Furthermore, those T cells (tetramers) for the identification and enumeration of antigen-
that recognized MHC I antigens invariably expressed the CD8 specific CD8+ T cells.271 In the horse, tetramers based on the
coreceptor. The nature of the association between MHC I and equine MHC I molecule 7-6 (Eqca-N*00602), associated with
the foreign antigen remained unclear until x-ray crystallo- the ELA-A1 haplotype, have been used to identify and quan-
graphic studies of human MHC I antigen were performed.269 tify equine infectious anemia virus (EIAV)-specific cytotoxic
In addition to revealing the structural organization of the T cells.272,273 Similar approaches are in development to analyze
domains of the MHC I antigen, the image also revealed a cleft CTL responses against other viruses and provide important
that lay between the α1 and α2 domains. It was proposed that information on the role these cells play in protection from
this cleft binds the processed peptide epitopes for presentation these infections.
to the T-cell receptor. Indeed, the cleft of the crystalized pro- Peptides that bind in the MHC I binding cleft are typically
tein used for the x-ray diffraction studies was found to contain 8 to 9 amino acids (aa) in length. Interestingly, three EIAV-
a contaminating peptide.269 Other experiments showed that specific CTL epitope peptides that bind to two closely related
the incubation of cells with purified viral peptides resulted equine MHC I molecules associated with ELA-A1 haplotype
in the lysis of the cells by virus-specific MHC I–restricted are 11 to 12 aa long.274 The two MHC I molecules, 7-6 (Eqca-
CTL.270 It is now known that the endogenous processing of N*00602) and 141 (Eqca-N*00601), differ in only one aa in the
viral antigens leads to the association of the viral peptides with α2 domain, yet present peptides to CTL differently, resulting
MHC I antigens on the surface of the infected cell, and this is in differential CTL recognition. Molecular modeling suggests
recognized by the TCR in association with CD8. How these that these peptides bind in a bulged conformation and pro-
viral antigens get to the cell surface is the result of a peptide vides an explanation for the experimentally observed differ-
transport system whose function is to transport processed ential recognition by CTL.274 Recently an independent group
peptides from the cytosol to the endoplasmic reticulum (ER). solved the crystal structures of these same MHC I molecules
Foreign and other cytosolic proteins are marked for degrada- with the bound peptides.275 This work confirmed the previ-
tion by covalent linkage to a small polypeptide called ubiqui- ous molecular modeling conclusions274 and has provided the
tin. These ubiquinated proteins are targeted for entry into a first crystal structures of equine MHC I–peptide complexes. In
proteasome, a cylindrical complex that degrades the protein addition, a shorter peptide (9 aa) with similar anchor residues
into small peptides. These are then transported into the ER bound the clefts of these molecules in a more typical confor-
by the transporter associated with antigen processing (TAP). mation.275 Despite MHC I binding, this 9 mer peptide may not
be a CTL epitope. The original study confirmed experimen- molecules play in antigen presentation to helper T cells (see
tally that although 1 of the 12 mer peptides bound the Eqca- later discussion), susceptibility to some diseases is associated
N*00601 molecule, there was no CTL recognition.274 Recent with particular MHC II haplotypes. For example, analysis
work with the Eqca-1*00101 MHC I molecule (associated of microsatellite markers and direct sequencing has demon-
with the ELA-A3 haplotype) has further defined equine MHC strated that the MHC II haplotype is a risk factor for insect bite
I peptide binding motifs and has demonstrated a narrow pep- hypersensitivity in defined populations of horses.286
tide binding repertoire.276 Moreover, CD8+ T-cell recognition Whereas intracellular antigens are processed via the endog-
of a large panel of EHV-1 peptides that bound this molecule enous pathway and are associated with MHC I molecules that
was restricted to a single 9 mer peptide. It will be of interest to present them to CD8+ cytotoxic T cells, extracellular antigens
determine whether any other equine MHC I molecules share are processed via the exogenous pathway and are associated
the limited peptide binding and TCR epitope recognition with MHC II molecules that present them to CD4+ helper
properties of Eqca-1*00101, as this would have implications T cells (Fig. 1.13). Here endocytosed antigens, such as that
for the control of intracellular infections in horses with some phagocytosed by a macrophage, are degraded into peptide
MHC I haplotypes. fragments within a phagolysosome compartment. Processed
MHC II molecules are heterodimeric, transmembrane gly- peptides then bind within the peptide binding cleft at the junc-
coproteins composed of an α chain and a β chain (see Fig. tion of the α1 and β1 domains of the MHC II molecule. This
1.12). A third chain, the invariant chain, has been shown to association of the epitope with the MHC II molecule protects
be associated with the MHC II molecule during assembly in it from further degradation. The MHC II molecule is then
the endoplasmic reticulum but is not expressed on the cell transported and expressed on the cell surface for subsequent
surface. Both the α and β polypeptides are encoded within presentation to the helper T cell. The immune system contains
the MHC region. Both polypeptides possess cytoplasmic, a distinct group of professional antigen-presenting cells called
transmembrane, and two extracellular domains (α1, α2 and dendritic cells that are specialized to capture antigens and ini-
β1, β2). The α1 and β1 domains comprise the peptide bind- tiate T-cell immunity and move freely from epithelial surfaces
ing cleft. The α chain has a single disulfide bond located in to adjoining lymph nodes. Dendritic cells can be found in a
its membrane proximal (α2) domain, and the β chain has a variety of locations in the body and are often named based
disulfide bond in both of its extracellular domains. Structur- on their microscopic appearance. Thus interdigitating cells
ally, the MHC II molecules resemble MHC I antigens and are found in lymph nodes, veiled cells in lymphatics, and Lang-
also members of the immunoglobulin superfamily, a group of erhans cells in skin are all dendritic cells. Immature dendritic
proteins that have structural similarities to immunoglobulin cells take up antigens by micropinocytosis using their exten-
molecules, including conserved domains and variable antigen sive cellular processes or by receptor-mediated phagocytosis.
binding domains. This results in activation and migration to a regional lymph
The MHC II genes are functionally and structurally distinct node where antigen presentation to T lymphocytes occurs.
from the MHC I genes. Unlike MHC I molecules, MHC II mol- Dendritic cells in the spleen capture blood-borne antigens and
ecules are restricted in their expression to certain cells of the similarly become activated, presenting antigens to T cells in
immune system: B lymphocytes, dendritic cells, macrophages, the periarteriolar lymphatic sheaths within white pulp. Mature
and activated T lymphocytes of some species. Other cells may dendritic cells have high levels of MHC II expression on their
also express MHC II molecules after treatment with various surface. Although no longer phagocytic, they are extremely
cytokines.277-279 Interferon-γ, TNF-α, 1,25-dihydroxyvitamin- efficient stimulators of both MHC I– and MHC II–restricted
D3, and granulocyte-macrophage colony-stimulating factor T-cell responses in the draining lymph node or other second-
can induce MHC II molecule expression on monocytes and ary lymphatic tissue (Fig. 1.14).
macrophages and other cells. IL-4 enhances MHC II expres- In a complex antigen, certain epitopes are particularly
sion on B cells. A number of agents have been shown to effective at stimulating an antibody response. These immuno-
downregulate MHC II expression, including glucocorticoids, dominant epitopes are often located at exposed areas of the
prostaglandins, and α-fetoprotein. Similar to MHC I, MHC II antigen such as in polypeptide loops. These types of structures
expression is regulated at the transcriptional level. The regu- are often quite mobile and may allow for easier access to the
latory regions are quite different, however, and this probably antibody binding site. T-cell epitopes have been shown to pos-
accounts for the differences in tissue distribution for these two sess a particular structural characteristic in that they involve
MHC molecules. the formation of amphipathic helices. However, structure
Like the MHC I genes, the MHC II region contains poly- alone does not determine the immunogenicity of a particu-
morphic genes encoding multiple MHC II molecules. There lar antigen, and T-cell recognition of foreign antigen requires
are loci for genes encoding the α chain (including DRA and more than just the expression of the processed antigen on
DQA loci) and β chain (including DRB and DQB loci), with the surface of the antigen-presenting cell. Additional signals
multiple alleles at most loci. In most species there is limited provided by the antigen-presenting cell are also required for
diversity in the α chain genes, with the β chain genes being the activation of the T lymphocytes. Among these are signals
the most polymorphic. For example, only two expressed provided by other accessory molecules found on the antigen
alleles occur at a single DRA locus in humans, whereas there presenting cell and various cytokines present in the extracel-
are multiple DRB loci containing over 1400 expressed alleles. lular environment.
Although the horse also has a single DRA locus, horses have
greater DRA allelic diversity than any other species.280-282 In Signaling through Antigen-Specific
addition, there are multiple DQA, DRB, and DQB loci, each Receptors
with multiple alleles contributing to the level of MHC II The binding of a specific antigen by either the TCR of a T
diversity in equids.282-285 Because of the critical role MHC II cell or the BCR of a B cell results in an intracellular signaling
Phagocytosis of
CTL TH extracellular antigen
3 1
Extracytoplasmic
Intracytoplasmic
KEY
MHC I molecule plus
chaperone molecules
MHC I molecule plus peptide

fragment of intracellular antigen
b MHC II molecule plus

invariant chain
Endoplasmic reticulum
MHC II molecule plus peptide
fragment of extracellular antigen
a
FIG. 1.13 Antigen processing pathways. This figure depicts MHC I antigen presentation to the left of the
diagram, and MHC II antigen presentation to the right. In MHC I antigen presentation (a) peptides generated
by degradation of proteins in the cytoplasm are transported into the endoplasmic reticulum by TAP (b). In
this location, MHC I molecules anchored by calnexin bind the antigenic peptides, which allows release of the
MHC I–peptide complex and transport through the Golgi to the cell surface (c). In MHC II antigen presenta-
tion, antigen is taken up by phagocytosis (1) into the endosome compartment that fuses with lysosomes for
degradation. Vesicles containing MHC II molecules produced in the endoplasmic reticulum fuse with the en-
dosomes (2), and the MHC II molecules bind with the degraded peptides for transport to the cell surface (3).
The MHC II molecules are prevented from binding the endogenous peptides in the endoplasmic reticulum
by the presence of an invariant chain, which is lost in the acidic endosomal environment.
cascade that eventually leads to the production of various Costimulatory Signals

cytokines and the proliferation of the stimulated cell. T-cell In addition to the interaction of TCR-CD3, CD4/CD8, and
recognition of antigen involves the engagement of a TCR- MHC-peptide antigen complexes, other cell surface molecules
CD4 or TCR-CD8 complex with processed peptide bound by are involved in signaling pathways. Of greatest importance is
an MHC II or MHC I molecule (Fig. 1.15). The engagement the interaction of CD28 on the T cell with B7 (CD80/86) on
of the TCR with the appropriate MHC-peptide complex also the antigen-presenting cell (APC). In the absence of CD28-
results in the binding of CD4 (or CD8) to a dedicated region CD80/86 costimulation, T cells are rendered functionally
of the MHC II (or MHC I). In doing so the lck protein tyrosine inactive or anergic. On restimulation, anergic T cells fail to
kinase associated with the cytoplasmic tail of CD4 (or CD8) proliferate or to produce cytokines such as IL-2 (a cytokine
phosphorylates the cytoplasmic regions of the CD3 proteins required for T-cell proliferation). The induction of anergy can
(associated with the TCR) in regions known as immunorecep- be prevented by either the addition of exogenous IL-2 or, more
tor tyrosine–based activation motifs (ITAM). A series of sub- important, by interaction of the CD28 cell surface antigen with
sequent phosphorylation events ultimately leads to the release its ligands (CD80/86). Stimulation of CD28 appears to be nec-
of stored Ca from the endoplasmic reticulum. The increase in essary for subsequent intracellular signaling events following
intracellular Ca levels and the activation of protein kinase C TCR stimulation as CD28 cross-linking enhances various bio-
leads to the phosphorylation of various transcriptional factors chemical events triggered by TCR-mediated signaling. Other
that regulate the expression of the genes for various cytokines molecules, including the TNF-receptor family member CD40,
and/or their receptors. The process is subsequently downregu- regulate T-cell growth and cell death. The engagement of CD40
lated by various phosphatases that are recruited to and subse- on the T cell with its ligand, CD40L (on APC), promotes cell
quently dephosphorylate the CD3 ITAMs. A similar process survival and cell cycle progression. The binding of other mem-
occurs in B cells when their surface immunoglobulin receptor bers of this family, notably TNF-α, to their receptors on acti-
is cross-linked on binding to specific antigen. The reader is vated T cells typically results in the activation of a biochemical
referred elsewhere for additional information regarding intra- cascade of caspases that leads to apoptosis (programmed cell
cellular signaling in lymphocytes.207,209 death). The cytocidal activity of these receptors is the result of
Pathogen invasion
Lymph node
Epithelial
surface
Paracortex
Follicle (T cell zone)
Dendritic cell Activation (B cell zone)
and migration
Antigen processing
and presentation
CTL
TH
FIG. 1.14 The role of professional antigen-presenting cells (APCs). In this figure pathogen invasion is fol-
lowed by antigen uptake by a dendritic cell, the most potent of the APC family. The dendritic cells become
activated and migrate to a local lymph node where they are extremely effective at stimulating naive T cells
in the paracortex, including both T-helper cells and CTLs.
TH CTL
T-cell receptor
Antigen
CD4 CD8
MHC II MHC I
Antigen presenting cell
FIG. 1.15 MHC class I and class II restricted T cell recognition: the role of T-cell CD4 and CD8 molecules.
T cells use their TCRs to recognize processed antigen presented in combination with either MHC I or MHC
II molecules. T cells exclusively express either CD4 (T-helper cells) or CD8 (cytotoxic T lymphocytes [CTLs]).
The CD4 molecule is required for interaction with MHC II molecules, and CD8 is required for MHC I interac-
tion. As a result, helper T cells only recognize antigen presented by MHC II molecules, and CTLs only recog-
nize antigen presented by MHC I molecules.
their intracytoplasmic portion of the receptor containing death Cytokines and Cell-Mediated Immunity
effector domains. By contrast, CD40 lacks the intracellular As indicated previously, cytokines are hormone-like proteins
death domains and instead has amino acid motifs that promote that mediate a variety of cellular responses. Over 100 differ-
activation. In addition to their role in promoting T-cell activa- ent cytokines and chemokines are known, and the reader is
tion and growth, both the CD28-CD80/86 and TNF-receptor referred elsewhere for comprehensive lists of their designa-
pathways may also play a dominant role in the induction of spe- tions and functions.207,209 Table 1.5 provides an abbreviated
cific T-helper cell subsets (see later discussion). list of cytokines with critical roles in innate and adaptive
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actors. Conversation would falter, then die down completely, her
presence would dry up their springs of inspiration. She spoilt things
while trying to make herself agreeable; they really liked her better
when she was grumpy.
Could Stephen have met men on equal terms, she would always
have chosen them as her companions; she preferred them because of
their blunt, open outlook, and with men she had much in common—
sport for instance. But men found her too clever if she ventured to
expand, and too dull if she suddenly subsided into shyness. In
addition to this there was something about her that antagonized
slightly, an unconscious presumption. Shy though she might be, they
sensed this presumption; it annoyed them, it made them feel on the
defensive. She was handsome but much too large and unyielding both
in body and mind, and they liked clinging women. They were oak-
trees, preferring the feminine ivy. It might cling rather close, it might
finally strangle, it frequently did, and yet they preferred it, and this
being so, they resented Stephen, suspecting something of the acorn
about her.
Stephen’s worst ordeals at this time were the dinners given in turn by
a hospitable county. They were long, these dinners, overloaded with
courses; they were heavy, being weighted with polite conversation;
they were stately, by reason of the family silver; above all they were
firmly conservative in spirit, as conservative as the marriage service
itself, and almost as insistent upon sex distinction.
‘Captain Ramsay, will you take Miss Gordon in to dinner?’
A politely crooked arm: ‘Delighted, Miss Gordon.’
Then the solemn and very ridiculous procession, animals marching
into Noah’s Ark two by two, very sure of divine protection—male and
female created He them! Stephen’s skirt would be long and her foot
might get entangled, and she with but one free hand at her disposal—
the procession would stop and she would have stopped it! Intolerable
thought, she had stopped the procession!
‘I’m so sorry, Captain Ramsay!’
‘I say, can I help you?’
‘No—it’s really—all right, I think I can manage—’
But oh, the utter confusion of spirit, the humiliating feeling that
some one must be laughing, the resentment at having to cling to his
arm for support, while Captain Ramsay looked patient.
‘Not much damage, I think you’ve just torn the frill, but I often
wonder how you women manage. Imagine a man in a dress like that,
too awful to think of—imagine me in it!’ Then a laugh, not unkindly but
a trifle self-conscious, and rather more than a trifle complacent.
Safely steered to her seat at the long dinner-table, Stephen would
struggle to smile and talk brightly, while her partner would think: ‘Lord,
she’s heavy in hand; I wish I had the mother; now there’s a lovely
woman!’
And Stephen would think: ‘I’m a bore, why is it?’ Then, ‘But if I
were he I wouldn’t be a bore, I could just be myself, I’d feel perfectly
natural.’
Her face would grow splotched with resentment and worry; she
would feel her neck flush and her hands become awkward.
Embarrassed, she would sit staring down at her hands, which would
seem to be growing more and more awkward. No escape! No escape!
Captain Ramsay was kind-hearted, he would try very hard to be
complimentary; his grey eyes would try to express admiration, polite
admiration as they rested on Stephen. His voice would sound softer
and more confidential, the voice that nice men reserve for good
women, protective, respectful, yet a little sex-conscious, a little
expectant of a tentative response. But Stephen would feel herself
growing more rigid with every kind word and gallant allusion. Openly
hostile she would be feeling, as poor Captain Ramsay or some other
victim was manfully trying to do his duty.
In such a mood as this she had once drunk champagne, one glass
only, the first she had ever tasted. She had gulped it all down in sheer
desperation—the result had not been Dutch courage but hiccups.
Violent, insistent, incorrigible hiccups had echoed along the whole
length of the table. One of those weird conversational lulls had been
filled, as it were, to the brim with her hiccups. Then Anna had started
to talk very loudly; Mrs. Antrim had smiled and so had their hostess.
Their hostess had finally beckoned to the butler: ‘Give Miss Gordon a
glass of water,’ she had whispered. After that Stephen shunned
champagne like the plague—better hopeless depression, she decided,
than hiccups!
It was strange how little her fine brain seemed able to help her
when she was trying to be social; in spite of her confident boasting to
Raftery, it did not seem able to help her at all. Perhaps is was the
clothes, for she lost all conceit the moment she was dressed as Anna
would have her; at this period clothes greatly influenced Stephen,
giving her confidence or the reverse. But be that as it might, people
thought her peculiar, and with them that was tantamount to
disapproval.
And thus, it was being borne in upon Stephen, that for her there
was no real abiding city beyond the strong, friendly old gates of
Morton, and she clung more and more to her home and to her father.
Perplexed and unhappy she would seek out her father on all social
occasions and would sit down beside him. Like a very small child this
large muscular creature would sit down beside him because she felt
lonely, and because youth most rightly resents isolation, and because
she had not yet learnt her hard lesson—she had not yet learnt that the
loneliest place in this world is the no-man’s-land of sex.
CHAPTER 9
S ir Philip and his daughter had a new common interest; they could
now discuss books and the making of books and the feel and the
smell and the essence of books—a mighty bond this, and one full of
enchantment. They could talk of these things with mutual
understanding; they did so for hours in the father’s study, and Sir
Philip discovered a secret ambition that had lain in the girl like a seed
in deep soil; and he, the good gardener of her body and spirit, hoed
the soil and watered this seed of ambition. Stephen would show him
her queer compositions, and would wait very breathless and still while
he read them; then one evening he looked up and saw her
expression, and he smiled:
‘So that’s it, you want to be a writer. Well, why not? You’ve got
plenty of talent, Stephen; I should be a proud man if you were a
writer.’ After which their discussions on the making of books held an
even more vital enchantment.
But Anna came less and less often to the study, and she would be
sitting alone and idle. Puddle, upstairs at work in the schoolroom,
might be swatting at her Greek to keep pace with Stephen, but Anna
would be sitting with her hands in her lap in the vast drawing-room so
beautifully proportioned, so restfully furnished in old polished walnut,
so redolent of beeswax and orris root and violets—all alone in its
vastness would Anna be sitting, with her white hands folded and idle.
A lovely and most comfortable woman she had been, and still was,
in spite of her gentle ageing, but not learned, oh, no, very far from
learned—that, indeed, was why Sir Philip had loved her, that was why
he had found her so infinitely restful, that was why he still loved her
after very many years; her simplicity was stronger to hold him than
learning. But now Anna went less and less often to the study.
It was not that they failed to make her feel welcome, but rather that
they could not conceal their deep interest in subjects of which she
knew little or nothing. What did she know of or care for the Classics?
What interest had she in the works of Erasmus? Her theology needed
no erudite discussion, her philosophy consisted of a home swept and
garnished, and as for the poets, she liked simple verses; for the rest
her poetry lay in her husband. All this she well knew and had no wish
to alter, yet lately there had come upon Anna an aching, a tormenting
aching that she dared give no name to. It nagged at her heart when
she went to that study and saw Sir Philip together with their daughter,
and knew that her presence contributed nothing to his happiness
when he sat reading to Stephen.
Staring at the girl she would see the strange resemblance, the
invidious likeness of the child to the father, she would notice their
movements so grotesquely alike; their hands were alike, they made
the same gestures, and her mind would recoil with that nameless
resentment, the while she reproached herself, penitent and trembling.
Yet penitent and trembling though Anna might be, she would
sometimes hear herself speaking to Stephen in a way that would
make her feel secretly ashamed. She would hear herself covertly,
cleverly gibing, with such skill that the girl would look up at her
bewildered; with such skill that even Sir Philip himself could not well
take exception to what she was saying; then, as like as not, she would
laugh if off lightly, as though all the time she had only been jesting,
and Stephen would laugh too, a big, friendly laugh. But Sir Philip
would not laugh, and his eyes would seek Anna’s, questioning,
amazed, incredulous and angry. That was why she now went so
seldom to the study when Sir Philip and his daughter were together.
But sometimes, when she was alone with her husband, Anna
would suddenly cling to him in silence. She would hide her face
against his hard shoulder clinging closer and closer, as though she
were frightened, as though she were afraid for this great love of theirs.
He would stand very still, forbearing to move, forbearing to question,
for why should he question? He knew already, and she knew that he
knew. Yet neither of them spoke it, this most unhappy thing, and their
silence spread round them like a poisonous miasma. The spectre that
was Stephen would seem to be watching, and Sir Philip would gently
release himself from Anna, while she, looking up, would see his tired
eyes, not angry any more, only very unhappy. She would think that
those eyes were pleading, beseeching; she would think: ‘He’s
pleading with me for Stephen.’ Then her own eyes would fill with tears
of contrition, and that night she would kneel long in prayer to her
Maker:
‘Give me peace,’ she would entreat, ‘and enlighten my spirit, so
that I may learn how to love my own child.’
Sir Philip looked older now than his age, and seeing this, Anna could
scarcely endure it. Everything in her cried out in rebellion so that she
wanted to thrust back the years, to hold them at bay with her own
weak body. Had the years been an army of naked swords she would
gladly have held them at bay with her body. He would constantly now
remain in his study right into the early hours of the morning. This habit
of his had been growing on him lately, and Anna, waking to find herself
alone, and feeling uneasy would steal down to listen. Backwards and
forwards, backwards and forwards! She would hear his desolate
sounding footsteps. Why was he pacing backwards and forwards, and
why was she always afraid to ask him? Why was the hand she
stretched out to the door always fearful when it came to turning the
handle? Oh, but it was strong, this thing that stood between them,
strong with the strength of their united bodies. It had drawn its own life
from their youth, their passion, from the splendid and purposeful
meaning of their passion—that was how it had leapt full of power into
life, and now it had thrust in between them. They were ageing, they
had little left but their loving—that gentler loving, perhaps the more
perfect—and their faith in each other, which was part of that loving,
and their peace, which was part of the peace of Morton. Backwards
and forwards, backwards and forwards! Those incessant and desolate
sounding footsteps. Peace? There was surely no peace in that study,
but rather some affliction, menacing, prophetic! Yet prophetic of what?
She dared not ask him, she dared not so much as turn the door-
handle, a haunting premonition of disaster would make her creep
away with her question unasked.
Then something would draw her, not back to her bedroom, but on
up the stairs to the room of their daughter. She would open that door
very gently—by inches. She would hold her hand so that it shaded the
candle, and would stand looking down at the sleeping Stephen as she
and her husband had done long ago. But now there would be no little
child to look down on, no small helplessness to arouse mother-pity.
Stephen would be lying very straight, very large, very long,
underneath the neatly drawn covers. Quite often an arm would be
outside the bedspread, the sleeve having fallen away as it lay there,
and that arm would look firm and strong and possessive, and so would
the face by the light of the candle. She slept deeply. Her breathing
would be even and placid. Her body would be drinking in its fill of
refreshment. It would rise up clean and refreshed in the morning; it
would eat, speak, move—it would move about Morton. In the stables,
in the gardens, in the neighbouring paddocks, in the study—it would
move about Morton. Intolerable dispensation of nature, Anna would
stare at that splendid young body, and would feel, as she did so, that
she looked on a stranger. She would scourge her heart and her
anxious spirit with memories drawn from this stranger’s beginnings:
‘Little—you were so very little!’ she would whisper, ‘and you sucked
from my breast because you were hungry—little and always so terribly
hungry—a good baby though, a contented little baby—’
And Stephen would sometimes stir in her sleep as though she
were vaguely conscious of Anna. It would pass and she would lie quiet
again, breathing in those deep, placid draughts of refreshment. Then
Anna, still ruthlessly scourging her heart and her anxious spirit, would
stoop and kiss Stephen, but lightly and very quickly on the forehead,
so that the girl should not be awakened. So that the girl should not
wake and kiss back, she would kiss her lightly and quickly on the
forehead.
The eye of youth is very observant. Youth has its moments and keen
intuition, even normal youth—but the intuition of those who stand
midway between the sexes, is so ruthless, so poignant, so accurate,
so deadly, as to be in the nature of an added scourge; and by such an
intuition did Stephen discover that all was not well with her parents.
Their outward existence seemed calm and unruffled; so far nothing
had disturbed the outward peace of Morton. But their child saw their
hearts with the eyes of the spirit; flesh of their flesh, she had sprung
from their hearts, and she knew that those hearts were heavy. They
said nothing, but she sensed that some deep, secret trouble was
afflicting them both; she could see it in their eyes. In the words that
they left unspoken she could hear it—it would be there, filling the small
gaps of silence. She thought that she discerned it in her father’s slow
movements—surely his movements had grown slower of late? And his
hair was quite grey; it was quite grey all over. She realized this with a
slight shock one morning as he sat in the sunlight—it had used to look
auburn in the nape of his neck when the sun fell upon it—and now it
was dull grey all over.
But this mattered little. Even their trouble mattered little in
comparison with something more vital, with their love—that, she felt,
was the only thing that mattered, and that was the thing that now
stood most in danger. This love of theirs had been a great glory; all
her life she had lived with it side by side, but never until it appeared to
be threatened, did she feel that she had really grasped its true
meaning—the serene and beautiful spirit of Morton clothed in flesh,
yes, that had been its true meaning. Yet that had been only part of its
meaning for her, it had meant something greater than Morton, it had
stood for the symbol of perfect fulfilment—she remembered that even
as a very small child she had vaguely discerned that perfect fulfilment.
This love had been glowing like a great friendly beacon, a thing that
was steadfast and very reassuring. All unconscious, she must often
have warmed herself at it, must have thawed out her doubts and her
vague misgivings. It had always been their love, the one for the other;
she knew this, and yet it had been her beacon. But now those flames
were no longer steadfast; something had dared to blemish their
brightness. She longed to leap up in her youth and strength and cast
this thing out of her holy of holies. The fire must not die and leave her
in darkness.
And yet she was utterly helpless, and she knew it. All that she did
seemed inadequate and childish: ‘When I was a child I spake as a
child, I understood as a child, I thought as a child.’ Remembering
Saint Paul, she decided grimly that surely she had remained as a
child. She could sit and stare at them—these poor, stricken lovers—
with eyes that were scared and deeply reproachful: ‘You must not let
anything spoil your loving, I need it,’ her eyes could send them that
message. She could love them in her turn, possessively, fiercely:
‘You’re mine, mine, mine, the one perfect thing about me. You’re one
and you’re mine, I’m frightened, I need you!’ Her thoughts could send
them that message. She could start to caress them, awkwardly, shyly,
stroking their hands with her strong, bony fingers—first his hand, then
hers, then perhaps both together, so that they smiled in spite of their
trouble. But she dared not stand up before them accusing, and say:
‘I’m Stephen, I’m you, for you bred me. You shall not fail me by failing
yourselves. I’ve a right to demand that you shall not fail me!’ No, she
dared not stand up and speak such words as these—she had never
demanded anything from them.
Sometimes she would think them quietly over as two fellow
creatures whom chance had made her parents. Her father, her mother
—a man, a woman; and then she would be amazed to discover how
little she knew of this man and this woman. They had once been
babies, and later small children, ignorant of life and utterly dependent.
That seemed so curious, ignorant of life—her father utterly weak and
dependent. They had come to adolescence even as she had, and
perhaps at times they too had felt unhappy. What had their thoughts
been, those thoughts that lie hidden, those nebulous misgivings that
never get spoken? Had her mother shrunk back resentful, protesting,
when the seal of her womanhood had been stamped upon her? Surely
not, for her mother was somehow so perfect, that all that befell her
must in its turn, be perfect—her mother gathered nature into her arms
and embraced it as a friend, as a well loved companion. But she,
Stephen, had never felt friendly like that, which must mean, she
supposed, that she lacked some fine instinct.
There had been those young years of her mother’s in Ireland; she
spoke of them sometimes but only vaguely, as though they were now
very far away, as though they had never seriously counted. And yet
she had been lovely, lovely Anna Molloy, much admired, much loved
and constantly courted—And her father, he too had been in the world,
in Rome, in Paris, and often in London—he had not lived much at
Morton in those days; and how queer it seemed, there had been a
time when her father had actually not known her mother. They had
been completely unconscious of each other, he for twenty-nine years,
she for just over twenty, and yet all the while had been drawing
together, in spite of themselves, always nearer together. Then had
come that morning away in County Clare, when those two had
suddenly seen each other, and had known from that moment the
meaning of life, of love, just because they had seen each other. Her
father spoke very seldom of such things, but this much he had told
her, it had all grown quite clear—What had it felt like when they
realized each other? What did it feel like to see things quite clearly, to
know the innermost reason for things?
Morton—her mother had come home to Morton, to wonderful,
gently enfolding Morton. She had passed for the first time through the
heavy white doorway under the shining semi-circular fanlight. She had
walked into the old square hall with its bearskins, and its pictures of
funny, dressed-up looking Gordons—the hall with the whip-rack where
Stephen kept her whips—the hall with the beautiful iridescent window,
that looked over the lawns and herbaceous borders. Then, perhaps
hand in hand, they had passed beyond the hall, her father a man, her
mother a woman, with their destiny already upon them—and that
destiny of theirs had been Stephen.
Ten years. For ten years they had just had each other, each other
and Morton—surely wonderful years. But what had they been thinking
about all those years? Had they perhaps thought a little about
Stephen? Oh, but what could she hope to know of these things, their
thoughts, their feelings, their secret ambitions—she, who had not even
been conceived, she, who had not yet come into existence? They had
lived in a world that her eyes had not looked on; days and nights had
slipped into the weeks, months and years. Time had existed, but she,
Stephen, had not. They had lived through that time; it had gone to
their making; their present had been the result of its travail, had
sprung from its womb as she from her mother’s, only she had not
been a part of that travail, as she had been a part of her mother’s.
Hopeless! And yet she must try to know them, these two, every inch of
their hearts, of their minds; and knowing them, she must then try to
guard them—but him first, oh, him first—she did not ask why, she only
knew that because she loved him as she did, he would always have to
come first. Love was simply like that; it just followed its impulse and
asked no questions—it was beautifully simple. But for his sake she
must also love the thing that he loved, her mother, though this love
was somehow quite different; it was less hers than his, he had thrust it
upon her; it was not an integral part of her being. Nevertheless it too
must be served, for the happiness of one was that of the other. They
were indivisible, one flesh, one spirit, and whatever it was that had
crept in between them was trying to tear asunder this oneness—that
was why she, their child, must rise up and help them if she could, for
was she not the fruit of their oneness?
4
There were times when she would think that she must have been
mistaken, that no trouble was overshadowing her father; these would
be when they two were sitting in his study, for then he would seem
contented. Surrounded by his books, caressing their bindings, Sir
Philip would look carefree again and light-hearted.
‘No friends in the world like books,’ he would tell her. ‘Look at this
fellow in his old leather jacket!’
There were times, too, out hunting when he seemed very young,
as Raftery had been that first season. But the ten-year-old Raftery
was now wiser than Sir Philip, who would often behave like a
foolhardy schoolboy. He would give Stephen leads over hair-raising
places, and then, she safely landed, turn round and grin at her. He
liked her to ride the pick of his hunters these days, and would slyly
show off her prowess. The sport would bring back the old light to his
eyes, and his eyes would look happy as they rested on his daughter.
She would think: ‘I must have been terribly mistaken,’ and would
feel a great peace surge over her spirit.
He might say, as they slowly jogged home to Morton: ‘Did you
notice my youngster here take that stiff timber? Not bad for a five-
year-old, he’ll do nicely.’ And perhaps he might add: ‘Put a three on
that five, and then tell your old sire that he’s not so bad either! I’m fifty-
three, Stephen, I’ll be going in the wind if I don’t knock off smoking
quite soon, and that’s certain!’
Then Stephen would know that her father felt young, very young,
and was wanting her to flatter him a little.
But this mood would not last; it had often quite changed by the
time that the two of them reached the stables. She would notice with a
sudden pain in her heart that he stooped when he walked, not much
yet, but a little. And she loved his broad back, she had always loved it
—a kind, reassuring protective back. Then the thought would come
that perhaps its great kindness had caused it to stoop as though
bearing a burden; and the thought would come: ‘He is bearing a
burden, not his own, it’s some one else’s—but whose?’
CHAPTER 10
C hristmas came and with it the girl’s eighteenth birthday, but the
shadows that clung round her home did not lessen; nor could
Stephen, groping about in those shadows, find a way to win through to
the light. Every one tried to be cheerful and happy, as even sad people
will do at Christmas, while the gardeners brought in huge bundles of
holly with which to festoon the portraits of Gordons—rich, red-berried
holly that came from the hills, and that year after year would be sent
down to Morton. The courageous-eyed Gordons looked out from their
wreaths unsmiling, as though they were thinking of Stephen.
In the hall stood the Christmas-tree of her childhood, for Sir Philip
loved the old German custom which would seem to insist that even
the aged be as children and play with God on His birthday. At the top
of the tree swung the little wax Christ-child in His spangled nightgown
with gold and blue ribbons; and the little wax Christ-child bent
downwards and sideways because, although small, He was rather
heavy—or, as Stephen had thought when she too had been small,
because He was trying to look for His presents.
In the morning they all went to church in the village, and the church
smelt of coldness and freshly bruised greenstuff—of the laurel and
holly and pungent pine branches, that wreathed the oak pulpit and
framed the altar; and the anxious-faced eagle who must carry the
Scriptures on his wings, he too was looking quite festive. Very redolent
of England it was, that small church, with its apple-cheeked choirboys
in newly washed garments; with its young Oxford parson who in
summer played cricket to the glory of God and the good of the county;
with its trim congregation of neighbouring gentry who had recently
purchased an excellent organ, so that now they could hear the
opening bars of the hymns with a feeling of self-satisfaction, but with
something else too that came nearer to Heaven, because of those
lovely old songs of Christmas. The choir raised their sexless,
untroubled voices: ‘While shepherds watched their flocks . . .’ sang the
choir; and Anna’s soft mezzo mingled and blended with her husband’s
deep boom and Puddle’s soprano. Then Stephen sang too for the
sheer joy of singing, though her voice at best was inclined to be
husky: ‘While shepherds watched their flocks by night,’ carolled
Stephen—for some reason thinking of Raftery.
After church the habitual Christmas greetings: ‘Merry Christmas.’
‘Merry Christmas.’ ‘Same to you, many of them!’ Then home to Morton
and the large mid-day dinner—turkey, plum pudding with its crisp
brandy butter, and the mince-pies that invariably gave Puddle
indigestion. Then dessert with all sorts of sweet fruits out of boxes,
crystallized fruits that made your hands sticky, together with fruit from
the Morton green-houses; and from somewhere that no one could
ever remember, the elegant miniature Lady-apples that you ate skins
and all in two bites if you were greedy.
A long afternoon spent in waiting for darkness when Anna could
light the Christmas-tree candles; and no ringing of bells to disturb the
servants, not until they must all file in for their presents which were
piled up high round the base of the tree on which Anna would light the
small candles. Dusk—draw the curtains, it was dark enough now, and
some one must go and fetch Anna the taper, but she must take care of
the little wax Christ-child, Who liked many lights even though they
should melt Him.
‘Stephen, climb up, will you, and tie back the Christ-child, His toe is
almost touching that candle!’
Then Anna applying the long lighted taper from branch to branch,
very slowly and gravely, as though she accomplished some ritual, as
though she herself were a ministering priestess—Anna very slender
and tall in a dress whose soft folds swept her limbs and lay round her
ankles.
‘Ring three times, will you, Philip? I think they’re all lighted—no,
wait—all right now, I’d missed that top candle. Stephen, begin to sort
out the presents, please, dear, your father’s just rung for the servants.
Oh, and Puddle, you might push over the table, I may need it—no, not
that one, the table by the window—’
A subdued sound of voices, a stifled giggle. The servants filing in
through the green baize door, and only the butler and footmen familiar
in appearance, the others all strangers, in mufti. Mrs. Wilson, the
cook, in black silk with jet trimming, the scullery maid in electric blue
cashmere, one housemaid in mauve, another in green, and the upper
of three in dark terracotta, while Anna’s own maid wore an old dress of
Anna’s. Then the men from outside, from the gardens and stables—
men bareheaded who were usually seen in their caps—old Williams
displaying a widening bald patch, and wearing tight trousers instead of
his breeches; old Williams walking stiffly because his new suit felt like
cardboard, and because his white collar was too high, and because
his hard, made-up black bow would slip crooked. The grooms and the
boys, all exceedingly shiny from their neatly oiled heads to their well
polished noses—the boys very awkward, short-sleeved and rough-
handed, shuffling a little because trying not to. And the gardeners led
in by the grave Mr. Hopkins, who wore black of a Sunday and carried
a Church Service, and whose knowledge of the ills that all grape-flesh
is heir to, had given his face a patient, pained expression. Men
smelling of soil these, in spite of much scrubbing; men whose necks
and whose hands were crossed and re-crossed by a network of tiny
and earth-clogged furrows—men whose backs would bend early from
tending the earth. There they stood in the wake of the grave Mr.
Hopkins, with their eyes on the big, lighted Christmas-tree, while they
never so much as glanced at the flowers that had sprung from many
long hours of their labour. No, instead they must just stand and gape
at the tree, as though with its candles and Christ-child and all, it were
some strange exotic plant in Kew Gardens.
Then Anna called her people by name, and to each one she gave
the gifts of that Christmas; and they thanked her, thanked Stephen
and thanked Sir Philip; and Sir Philip thanked them for their faithful
service, as had always been the good custom at Morton for more
years than Sir Philip himself could remember. Thus the day had
passed by in accordance with tradition, every one from the highest to
the lowest remembered; nor had Anna forgotten her gifts for the
village—warm shawls, sacks of coal, cough mixture and sweets. Sir
Philip had sent a cheque to the vicar, which would keep him for a long
time in cricketing flannels; and Stephen had carried a carrot to Raftery
and two lumps of sugar to the fat, aged Collins, who because he was
all but blind of one eye, had bitten her hand in place of his sugar. And
Puddle had written at great length to a sister who lived down in
Cornwall and whom she neglected, except on such memory-jogging
occasions as Christmas, when somehow we always remember. And
the servants had gorged themselves to repletion, and the hunters had
rested in their hay-scented stables; while out in the fields, sea-gulls,
come far inland, had feasted in their turn on humbler creatures—grubs
and slugs, and other unhappy small fry, much relished by birds and
hated by farmers.
Night closed down on the house, and out of the darkness came the
anxious young voices of village schoolchildren: ‘Noël, Noël—’ piped
the anxious young voices, lubricated by sweets from the lady of
Morton. Sir Philip stirred the logs in the hall to a blaze, while Anna
sank into a deep chair and watched them. Her hands that were
wearied by much ministration, lay over the arms of the chair in the
firelight, and the firelight sought out the rings on her hands, and it
played with the whiter flames in her diamonds. Then Sir Philip stood
up, and he gazed at his wife, while she stared at the logs not
appearing to notice him; but Stephen, watching in silence from her
corner, seemed to see a dark shadow that stole in between them—
beyond this her vision was mercifully dim, otherwise she must surely
have recognized that shadow.
On New Year’s Eve Mrs. Antrim gave a dance in order, or so she

said, to please Violet, who was still rather young to attend the hunt
balls, but who dearly loved gaiety, especially dancing. Violet was
plump, pert and adolescent, and had lately insisted on putting her hair
up. She liked men, who in consequence always liked her, for like
begets like when it comes to the sexes, and Violet was full of what
people call: ‘allure,’ or in simpler language, of sexual attraction. Roger
was home for Christmas from Sandiest, so that he would be there to
assist his mother. He was now nearly twenty, a good-looking youth
with a tiny moustache which he tentatively fingered. He assumed the
grand air of the man of the world who has actually weathered about
nineteen summers. He was hoping to join his regiment quite soon,
which greatly augmented his self-importance.
Could Mrs. Antrim have ignored Stephen Gordon’s existence, she
would almost certainly have done so. She disliked the girl; she had
always disliked her; what she called Stephen’s ‘queerness’ aroused
her suspicion—she was never quite clear as to what she suspected,
but felt sure that it must be something outlandish: ‘A young woman of
her age to ride like a man, I call it preposterous!’ declared Mrs. Antrim.
It can safely be said that Stephen at eighteen had in no way
outgrown her dread of the Antrims; there was only one member of that
family who liked her, she knew, and that was the small, hen-pecked
Colonel. He liked her because, a fine horseman himself, he admired
her skill and her courage out hunting.
‘It’s a pity she’s so tall, of course—’ he would grumble, ‘but she
does know a horse and how to stick on one. Now my children might
have been brought up at Margate, they’re just about fitted to ride the
beach donkeys!’
But Colonel Antrim would not count at the dance; indeed in his own
house he very seldom counted. Stephen would have to endure Mrs.
Antrim and Violet—and then Roger was home from Sandiest. Their
antagonism had never quite died, perhaps because it was too
fundamental. Now they covered it up with a cloak of good manners,
but these two were still enemies at heart, and they knew it. No,
Stephen did not want to go to that dance, though she went in order to
please her mother. Nervous, awkward and apprehensive, Stephen
arrived at the Antrims that night, little thinking that Fate, the most
expert of tricksters, was waiting to catch her just round the corner. Yet
so it was, for during that evening Stephen met Martin and Martin met
Stephen, and their meeting was great with portent for them both,
though neither of them could know it.
It all happened quite simply as such things will happen. It was
Roger who introduced Martin Hallam; it was Stephen who explained
that she danced very badly; it was Martin who suggested that they sit
out their dances. Then—how quickly it occurs if the thing is pre-
destined—they suddenly knew that they liked each other, that some
chord had been struck to a pleasant vibration; and this being so they
sat out many dances, and they talked for quite a long while that
evening.
Martin lived in British Columbia, it seemed, where he owned
several farms and a number of orchards. He had gone out there after
the death of his mother, for six months, but had stayed on for love of
the country. And now he was having a holiday in England—that was
how he had got to know young Roger Antrim, they had met up in
London and Roger had asked him to come down for a week, and so
here he was—but it felt almost strange to be back again in England.
Then he talked of the vastness of that new country that was yet so old;
of its snow-capped mountains, of its canyons and gorges, of its deep,
princely rivers, of its lakes, above all of its mighty forests. And when
Martin spoke of those mighty forests, his voice changed, it became
almost reverential; for this young man loved trees with a primitive
instinct, with a strange and inexplicable devotion. Because he liked
Stephen he could talk of his trees, and because she liked him she
could listen while he talked, feeling that she too would love his great
forests. His face was very young, clean-shaven and bony; he had
bony, brown hands with spatulate fingers; for the rest, he was tall with
a loosely knit figure, and he slouched a little when he walked, from
much riding. But his face had a charming quality about it, especially
when he talked of his trees; it glowed, it seemed to be inwardly
kindled, and it asked for a real and heart-felt understanding of the
patience and the beauty and the goodness of trees—it was eager for
your understanding. Yet in spite of this touch of romance in his make-
up, which he could not keep out of his voice at moments, he spoke
simply, as one man will speak to another, very simply, not trying to
create an impression. He talked about trees as some men talk of
ships, because they love them and the element they stand for. And
Stephen, the awkward, the bashful, the tongue-tied, heard herself
talking in her turn, quite freely, heard herself asking him endless
questions about forestry, farming and the care of vast orchards;
thoughtful questions, unromantic but apt—such as one man will ask of
another.
Then Martin wished to learn about her, and they talked of her
fencing, her studies, her riding, and she told him about Raftery who
was named for the poet. And all the while she felt natural and happy
because here was a man who was taking her for granted, who
appeared to find nothing eccentric about her or her tastes, but who
quite simply took her for granted. Had you asked Martin Hallam to
explain why it was that he accepted the girl at her own valuation, he
would surely have been unable to tell you—it had happened, that was
all, and there the thing ended. But whatever the reason, he felt drawn
to this friendship that had leapt so suddenly into being.
Before Anna left the dance with her daughter, she invited the
young man to drive over and see them; and Stephen felt glad of that
invitation, because now she could share her new friend with Morton.
She said to Morton that night in her bedroom: ‘I know you’re going to
like Martin Hallam.’
CHAPTER 11
M artin went to Morton, he went very often, for Sir Philip liked him
and encouraged the friendship. Anna liked Martin too, and she
made him feel welcome because he was young and had lost his
mother. She spoilt him a little, as a woman will spoil who, having no son
must adopt some one else’s, so to Anna he went with all his small
troubles, and she doctored him when he caught a bad chill out hunting.
He instinctively turned to her in such things, but never, in spite of their
friendship, to Stephen.
Yet now he and Stephen were always together, he was staying on
and on at the hotel in Upton; ostensibly staying because of the hunting;
in reality staying because of Stephen who was filling a niche in his life
long empty, the niche reserved for the perfect companion. A queer,
sensitive fellow this Martin Hallam, with his strange love of trees and
primitive forests—not a man to make many intimate friends, and in
consequence a man to be lonely. He knew little about books and had
been a slack student, but Stephen and he had other things in common;
he rode well, and he cared for and understood horses; he fenced well
and would quite often now fence with Stephen; nor did he appear to
resent it when she beat him; indeed he seemed to accept it as natural,
and would merely laugh at his own lack of skill. Out hunting these two
would keep close to each other, and would ride home together as far as
Upton; or perhaps he would go on to Morton with her for Anna was
always glad to see Martin. Sir Philip gave him the freedom of the
stables, and even old Williams forbore to grumble:
‘ ’E be trusty, that’s what ’e be,’ declared Williams, ‘and the horses
knows it and acts accordin’.’
But sport was not all that drew Stephen to Martin, for his mind, like
hers, was responsive to beauty, and she taught him the country-side
that she loved, from Upton to Castle Morton common—the common
that lies at the foot of the hills. But far beyond Castle Morton she took
him. They would ride down the winding lane to Bromsberrow, then
crossing the small stream at Clincher’s Mill, jog home through the bare
winter woods of Eastnor. And she taught him the hills whose plentiful
bosoms had made Anna think of green-girdled mothers, mothers of
sons, as she sat and watched them, great with the child who should
have been her son. They climbed the venerable Worcestershire
Beacon that stands guardian of all the seven Malverns, or wandered
across the hills of the Wells to the old British Camp above the Wye
Valley. The Valley would lie half in light, half in shadow, and beyond
would be Wales and the dim Black Mountains. Then Stephen’s heart
would tighten a little, as it always had done because of that beauty, so
that one day she said:
‘When I was a child, this used to make me want to cry, Martin.’
And he answered: ‘Some part of us always sheds tears when we
see lovely things—they make us regretful.’ But when she asked him
why this should be, he shook his head slowly, unable to tell her.
Sometimes they walked through Hollybush woods, then on up
Raggedstone, a hill grim with legend—its shadow would bring
misfortune or death to those it fell on, according to legend. Martin
would pause to examine the thorn trees, ancient thorns that had
weathered many a hard winter. He would touch them with gentle,
pitying fingers:
‘Look, Stephen—the courage of these old fellows! They’re all
twisted and crippled; it hurts me to see them, yet they go on patiently
doing their bit—have you ever thought about the enormous courage of
trees? I have, and it seems to me amazing. The Lord dumps them
down and they’ve just got to stick it, no matter what happens—that
must need some courage!’ And one day he said: ‘Don’t think me quite
mad, but if we survive death then the trees will survive it; there must be
some sort of a forest heaven for all the faithful—the faithful of trees. I
expect they take their birds along with them; why not? “And in death
they were not divided.” ’ Then he laughed, but she saw that his eyes
were quite grave, so she asked him:
‘Do you believe in God, Martin?’
And he answered: ‘Yes, because of His trees. Don’t you?’
‘I’m not sure—’

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Stephen M. Reed, DVM, Dipl ACVIM

Warwick M. Bayly, BVSc, MS, PhD, Dipl ACVIM

Debra C. Sellon, DVM, PhD, Dipl ACVIM

EQUINE INTERNAL MEDICINE ISBN: 978-0-323-44329-6

Copyright © 2018, Elsevier Inc. All rights reserved.

Library of Congress Cataloging-in-Publication Data

Senior Content Strategist: Jennifer Flynn-Briggs

Printed in United States of America

Last digit is the print number: 9 8 7 6 5 4 3 2 1

Robert H. Mealey, DVM, PhD, DACVIM

Patricia Talcott, DVM, MS, PhD, DABVT

Mechanisms of Disease and

nondisrupted skin surfaces. Even though horses inhabit an

TABLE 1.2 Major Types of Bacterial Adhesins

in a guinea pig model.93 Both of these mutants can proliferate

Coronaviridae Paramyxoviridae Bunyaviridae Toroviridae

RNA Orthomyxoviridae Arenaviridae Togaviridae Flaviridae ssRNA

Protein Synthetic Replicative New Viral

dsDNA Retroviridae dsDNA

1 (Fig. 1.5). Localized viral replication first occurs at the site of

1 Regional lymph node:

Skin: Focal infection

immunology.206 Work in the horse continues in many of Proinflammatory Cytokines,

Lectin Pathway Classical Pathway Alternate Pathway

Chemotaxis Opsonization Microbial Lysis

(NADPH)-oxidase–dependent respiratory burst. During Adaptive Immunity

CD4 TH1 IFN-γ

Distinguished by cell surface molecules Functional distinctions

Variable region Constant regions Fc region Fab region

TABLE 1.4 Immunoglobulin Isotypes

Ig gene rearrangement Antigen driven

cytoplasmic µ surface surface surface

Variable region genes Constant region genes

IgGa ? IgG(T) IgGb IgGc IgG(T)

V1 2 3 4 5 D1 J2 Cµ Cδ Cγ1 Cγ2 Cγ3 Cγ4 Cγ5 Cγ6 Cγ6 Cε Cα

V5 D1 J2 Cµ Cδ Cγ1 Cγ2 Cγ3 Cγ4 Cγ5 Cγ6 Cγ6 Cε Cα

Complete IgGb heavy chain polypeptide chain

Variable region Constant regions

Immature CD4-/CD8- double negative

Cortex Immature CD4+/CD8+ thymocytes interact

FIG. 1.11 T-cell maturation and selection in the thymus.

MHC I molecule plus peptide

b MHC II molecule plus

cascade that eventually leads to the production of various Costimulatory Signals

Antigen presenting cell

On New Year’s Eve Mrs. Antrim gave a dance in order, or so she

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EQUINE INTERNAL MEDICINE ISBN: 978-0-323-44329-6