Pathology of Wildlife and Zoo Animals

Pathology of Wildlife and Zoo Animals

Edited by

Karen A. Terio

University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Denise McAloose

Wildlife Conservation Society, Bronx Zoo, Bronx, NY, United States

Judy St. Leger

SeaWorld Parks and Entertainment, San Diego, CA, United States

Table of Contents

Cover

Title page

Copyright

Dedication

Contributors

Foreword

Preface

Chapter 1: Wildlife Necropsy

Abstract

Introduction

Necropsy basics

Necropsy examination

General

Arthropod necropsy evaluation

Cnidarian necropsy evaluation

Echinoderm necropsy evaluation

Bivalve and gastropod mollusc necropsy evaluation

Cephalopod mollusc necropsy evaluation

General necropsy examination form

Chapter 2: Forensic Wildlife Pathology

Abstract

Introduction

Trauma

Toxins

Ballistics

Drowning

Poisoning

Forensic taphonomy and time of death determination

Gross and histologic nonlesions in the frozen/thawed carcass

Skeletal preparation techniques

Documentation and reporting

Report content

Preservation of evidence

Chapter 3: Laboratory Diagnostics

Abstract

Principles of diagnostic testing

Choosing the right test

Reference data requirements

Test performance and interpretation

Using test results for decision making

Evidence for causality

Sample handling and diagnostics

Choosing a laboratory

New tools for pathogen discovery

Chapter 4: Introduction to Comparative Clinical Pathology

Abstract

Basic principles of clinical pathology across species

Comparative clinical pathology and unique clinical pathology features in nondomestic species

Chapter 5: Bovidae, Antilocapridae, Giraffidae, Tragulidae, Hippopotamidae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 6: Cervidae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 7: Camelidae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 8: Suidae and Tayassuidae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 9: Canidae, Ursidae, and Ailuridae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 10: Felidae

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Common neoplasms in felids

Chapter 11: Mustelids

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

Chapter 12: Procyonidae, Viverridae, Hyenidae, Herpestidae, Eupleridae, and Prionodontidae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

RNA Viruses

E-Slides

Chapter 13: Prosimians

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 14: New World and Old World Monkeys

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Classification of New World and Old World monkeys

Unique features

Additional notable infectious diseases

Chapter 15: Apes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Introduction to apes

Additional unique features

Additional viruses and viral diversity in apes

Additional parasites of apes

Chapter 16: Proboscidae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

Parasites

E-Slides

Chapter 17: Perissodactyls

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 18: Monotremes and Marsupials

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Unique anatomical features of monotremes and marsupials

Chapter 19: Lagomorpha

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 20: Rodentia

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 21: Xenartha, Erinacoemorpha, Some Afrotheria, and Phloidota

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 22: Cetacea

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 23: Pinnipediae

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 24: Sirenia

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Additional unique clinical and anatomic features

Unique features

Chapter 25: Chiroptera

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Additional toxic diseases

Additional bacterial diseases

Additional fungal diseases

Additional protozoal diseases

Chapter 26: Palaeognathae: Apterygiformes, Casuariiformes, Rheiformes, Struthioniformes; Tinamiformes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

General ratite management and health reference materials

Notable clinical pathology information and reference materials in ratites

References

Toxic elements described as causes of clinical disease and death in large ratite species

Chapter 27: Sphenisciformes, Gaviiformes, Podicipediformes, Procellariiformes, and Pelecaniformes

Abstract

Introduction

Unique features

Non-infectious diseases

Congenital/Genetic

Age-related/Degenerative

Inflammatory non-infectious

Infectious diseases

E-Slides

Chapter 28: Phoenicopteriformes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 29: Anseriformes, Ciconiiformes, Charadriiformes, and Gruiformes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Infectious diseases

Chapter 30: Birds of Prey

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 31: Galliformes and Columbiformes

Abstract

Introduction

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 32: Psittacines, Coliiformes, Musophagiformes, Cuculiformes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Introduction

Unique features

Non-infectious diseases

Infectious diseases

Chapter 33: Passeriformes, Caprimulgiformes, Coraciiformes, Piciformes, Bucerotiformes, and Apodiformes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 34: Chelonia

Abstract

Introduction

Unique features

Clinical pathology

Non-infectious diseases

Infectious diseases

E-Slides

Additional unique features

Hematology

Clinical chemistry

Chapter 35: Crocodilia

Abstract

Introduction

Non-infectious diseases

Infectious diseases

Presumed infectious disease with uncertain etiology

E-Slides

Additional unique features

Chapter 36: Lacertilia

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Lizard taxonomy

Chapter 37: Serpentes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 38: Amphibia

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Chapter 39: Osteichthyes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Additional unique features

Chapter 40: Chondrichthyes

Abstract

Introduction

Unique features

Non-infectious diseases

Infectious diseases

E-Slides

Additional unique features

Chapter 41: Invertebrates

Abstract

Introduction

Unique features

Multifactorial conditions

Non-infectious diseases

Infectious diseases

E-Slides

Additional unique features

Appendix A: Viral Families and Documented Diseases

Index

Copyright

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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.

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A catalogue record for this book is available from the British Library

ISBN: 978-0-12-805306-5

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Dedication

If I have seen further, it is by standing on the shoulders of giants

Sir Isaac Newton

(after Bernard of Chartres)

Dedicated to our mentors and colleagues who have and continue

to inspire us with their passion and expertise.

In memory of Dr. Richard (Dick) J. Montali and Dr. Linda Munson

Contributors

Dalen Agnew,     Michigan State University, Lansing, MI, United States

Anibal G. Armien,     University of Minnesota, St. Paul, MN, United States

Josephine Braun,     Disease Investigations, Institute for Conservation Research, San Diego Zoo Global, San Diego, CA, United States

Elizabeth L. Buckles,     Cornell University, Ithaca, NY, United States

Kathy A. Burek-Huntington,     Alaska Veterinary Pathology Services, Eagle River, AK, United States

Alvin C. Camus,     University of Georgia, Athens, GA, United States

María del Carmen Carmona Muciño,     Africam Zoo Safari Puebla, Mexico

Tylis Y. Chang,     Zucker School of Medicine, New Hyde Park, NY, United States

Molly E. Church,     University of Pennsylvania, Philadelphia, PA, United States

Kathleen M. Colegrove,     University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Kenneth J. Conley,     Wildlife Conservation Society, Bronx Zoo, Bronx, NY, United States

Rocio Crespo,     Washington State University, Puyallup, WA, United States

Martha A. Delaney,     University of Washington School of Medicine, Seattle, WA, United States

Daniela Denk,     International Zoo Veterinary Group, Keighley, West Yorkshire, United Kingdom

Martine de Wit,     Florida Fish and Wildlife Conservation Commission, Saint Petersburg, FL, United States

Gerry Dorrestein,     Pathology Laboratory NOIVBD, Vessem, The Netherlands

Mary Duncan,     St. Louis Zoo, St. Louis, MO, United States

Lisa L. Farina,     University of Florida, Gainesville, FL, United States

Heather Fenton,     University of Georgia, Athens, GA, United States

Mark Flint

The University of Queensland, Gatton, QLD, Australia

The Ohio State University, Columbus, OH, United States

Monique S. França,     Poultry Diagnostic and Research Center, The University of Georgia, Athens, GA, United States

Salvatore Frasca, Jr,     University of Connecticut, Storrs, CT, United States

Michael M. Garner,     Northwest ZooPath, Monroe, WA, United States

David J. Gasper,     Pacific Zoo & Wildlife Diagnostics, San Diego, CA, United States

Ana María Henao Duque,     Zoopath Veterinary Pathology Laboratory-Cali Zoo, Cali, Colombia

Damien Higgins,     The University of Sydney, Sydney, Australia

Ursula Höfle,     SaBio (Health and Biotechnology) Working Group, National Game and Wildlife Research Institute, Cuidad Real, Spain

Charlotte Hollinger,     Wildlife Conservation Society, Bronx Zoo, Bronx, NY, United States

Elizabeth W. Howerth,     University of Georgia, Athens, GA, United States

María Ángeles Jiménez Martínez,     Veterinary Faculty Complutense University of Madrid, Spain

Megan E.B. Jones,     Institute for Conservation Research, San Diego Zoo Global, San Diego, CA, United States

Carles Juan-Sallés,     Noah’s Path, Elche (Alicante), Spain

Rebecca A. Kagan,     United States Fish and Wildlife Service, Ashland, OR, United States

M. Kevin Keel,     University of California, Davis, CA, United States

Michael J. Kinsel,     University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Jörg Kinne,     Central Veterinary Research Laboratory, Dubai, United Arab Emirates

Jennifer A. Landolfi,     University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Julia S. Lankton,     U.S. Geological Survey-National Wildlife Health Center, Madison, WI, United States

Eric D. Lombardini,     Public Health Command-Fort District Carson, Fort Carson, CO, United States

Linda J. Lowenstine,     University of California, Davis, CA, United States

Kerstin Mätz-Rensing,     German Primate Center, Leibniz Institute of Primate Research, Göttingen, Germany

Denise McAloose,     Wildlife Conservation Society, Bronx Zoo, Bronx, NY, United States

Rita McManamon,     University of Georgia, Athens, GA, United States

Alexandria Mena,     SeaWorld Parks and Entertainment, San Diego, CA, United States

Melissa Miller,     Marine Wildlife Veterinary Care and Research Center, Santa Cruz, CA, United States

Emily Mitchell (née Lane)

National Zoological Gardens of South Africa

University of Pretoria, Pretoria, South Africa

Nicole M. Nemeth,     University of Guelph, Guelph, ON, Canada

Alisa L. Newton,     Wildlife Conservation Society, Bronx, NY, United States

Sally Nofs,     Potter Park Zoo, Lansing, MI, United States

Helen Owen,     The University of Queensland, Gatton, QLD, Australia

Francesco C. Origgi,     University of Bern, Centre for Fish and Wildlife Health-FIWI, Bern, Switzerland

Robert J. Ossiboff,     University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Allan P. Pessier,     Washington State University, Pullman, WA, United States

Stephen Raverty,     Animal Health Center, Abbottsford, BC, Canada

Drury R. Reavill,     Zoo/Exotic Pathology Service, Citrus Heights, CA, United States

Bruce Rideout,     Disease Investigations, Institute for Conservation Research, San Diego Zoo Global, San Diego, CA, United States

Carlos E. Rodriguez,     Walt Disney Parks and Resorts, Orlando, FL, United States

Wendi Roe,     Massey University, Palmerston North, New Zealand

Karrie Rose

Australian Registry of Wildlife Health, Taronga Conservation Society Australia, Mosman, NSW

The University of Sydney, Sydney, Australia

Jamie L. Rothenburger,     Ontario Veterinary College, University of Guelph, Guelph, ON, Canada

Marie-Pierre Ryser-Degiorgis,     Centre for Fish and Wildlife Health, University of Bern, Bern, Switzerland

H.L. Shivaprasad,     California Animal Health and Food Safety Laboratory System—Tulare Branch, University of California, Davis, Tulare, CA, United States

Catherine M. Shilton,     Northern Territory Department of Primary Industry and Resources, NT, Australia

Ursula Siebert,     University of Veterinary Medicine Hannover, Hannover, Germany

Dale A. Smith,     University of Guelph, Guelph, ON, Canada

Roxanna Smolowitz,     Roger Williams University, Bristol, RI, United States

David Spratt,     CSIRO, Australian National Wildlife Collection, Canberra, Australia

Judy St. Leger,     SeaWorld Parks and Entertainment, San Diego, CA, United States

Nicole I. Stacy,     University of Florida, Gainesville, FL, United States

Ilse H. Stalis,     Disease Investigations, Institute for Conservation Research, San Diego Zoo Global, San Diego, CA, United States

Nancy L. Stedman,     Busch Gardens Tampa, Tampa, FL, United States

Jennifer Steinberg,     IDEXX Reference Laboratories, Glen Burnie, MD, United States

Mark F. Stidworthy,     International Zoo Veterinary Group, Keighley, West Yorkshire, United Kingdom

Karen A. Terio,     University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Scott P. Terrell,     Walt Disney Parks and Resorts, Orlando, FL, United States

Piper M. Treuting,     University of Washington School of Medicine, Seattle, WA, United States

John Trupkiewicz,     Philadelphia Zoo, Philadelphia, PA, United States

Tabitha C. Viner,     United States Fish and Wildlife Service, Ashland, OR, United States

Bruce H. Williams,     Joint Pathology Center, Silver Spring, MD, United States

Jeffrey C. Wolf,     Experimental Pathology Laboratories, Inc., Sterling, VA, United States

Daniel B. Woodburn,     University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Arno Wünschmann,     University of Minnesota, St. Paul, MN, United States

Foreword

This book is an exciting, much needed, and extremely important addition to the veterinary literature. It is the first comprehensive compilation of the pathology of wild animals in managed care and natural habitats. It includes information about a wide range of taxa, from invertebrates to apes, and covers the field as we currently understand it in the second decade of the 21st century. The chapters are compilations of literature reviews and personal observations from veterinary pathologists with extensive expertise in the field. The author list includes a veritable Who’s Who of pathologists, although it is missing some of the most influential names in the field of wildlife and nondomestic animal pathology. Among these are Drs. Richard Montali, Rudolph Ippen, Beth Williams, and Linda Munson, who are, sadly, either no longer with us or are unable to participate due to illness. We owe a debt of gratitude to these and others whose contributions are reflected in the references cited and presence of their students as contributors throughout the book.

The aim of this book is to enhance the practice of evidence-based wildlife and zoo animal pathology by increasing the ability of veterinary pathologists to diagnose and interpret causes of morbidity and mortality in a diverse range of species, thus improving management and health of wild animals. Beyond veterinary pathologists, the text is designed to serve as a resource for students, trainees, veterinarians, biologists, and other scientists working with these animals. For many taxa, half the battle of making an accurate assessment of health and disease is to understand the range of normal gross and histological anatomy. Helpful anatomic hints are presented in the text, while more detail for several taxa is provided in the extensive on line supplemental materials, which also include much information on diseases not covered in the hard copy of the book. For many taxa, information on pathology is sparse. Other taxa, for example nonhuman primates, by virtue of biomedical research and curiosity about human origins, have been extensively studied. The field of nondomestic animal pathology has moved from anecdotal to science based case reports (which still continue to be necessary pieces of the jigsaw puzzle of our understanding), descriptive case series, and hypothesis driven investigations. Review papers, metaanalyses, case control studies, and laboratory research on pathogenesis at the cellular or molecular level are still relatively uncommon for most taxa.

Stepping back and looking at the discipline of wildlife and zoo pathology, we can find its roots in two different but complimentary areas. Disease investigations of free-ranging wild animals grew out of the field of wild game management and were often the purview of wildlife biologists working within a background of population biology, ecology, behavior, parasitology, and toxicology. In contrast, zoo animal pathology was initially practiced by zoologists with an interest in anatomy from the stand point of systematics and by medical doctors or pathologists curious about comparative aspects of diseases or searching for models of human diseases (or who may have been bored with dealing with a single species!). Only in the last few decades have these two been melded into a single discipline accepted as a legitimate field within veterinary pathology, and practiced by an increasing cadre of veterinary pathologists, many of whom have received postgraduate pathology training and board certification by the American, European, or Japanese Colleges of Veterinary Pathologists.

Certainly, there have been several previous books on diseases of wildlife and zoo animal diseases. References date back to German language texts in the late 1800s and later English language texts and translations, such as Herbert Fox’s Disease in Captive Wild Mammals and Birds (J.B. Lippincott Co, 1923), Reichenbach-Klinke and Elkan’s Principal Diseases of Lower Vertebrates (Academic Press, 1965), Parasitic Diseases of Wild Mammals (Iowa State University Press, 1st edition, 1971), Infectious and Parasitic Diseases of Wild Birds (Iowa State Press, first edition 1971), and Fowler’s Zoo and Wild Animal Medicine (WB Saunders, first edition 1978). These have included descriptions of pathology, but the focus has largely been on clinical aspects of disease, epidemiology, management, and care. Likewise, texts, such as Runndell’s Animal Pathology (Iowa State University Press 1944), Diseases of Poultry (first edition 1943, Iowa State University Press), Smith and Jones’s Veterinary Pathology (Lea and Febiger, first edition 1957), Jubb and Kennedy’s Pathology of Domestic Animals (Academic Press, first edition 1963), and Fish Pathology (Bailliere Tindall, first edition, 1975) and their subsequent editions, ushered in a better understanding of mammalian, avian, and piscine domestic animal pathology. However, the greater diversity of animal species including invertebrates (with more than 1.3 million extant species), fish (about 33,000 extant species), amphibians (approximately 7,300 species), reptiles (about 10,000 species), birds (approximately 10,400 species), and nondomestic mammals (about 5,500 species) were largely ignored. None the less, information on domestic animal pathology has allowed useful extrapolation to wild species with close phylogenetic relationships to domestics (e.g., galliforms, bovids, suids, equids, canids, and felids). Information more specific to pathology of single nondomestic or nonmammalian taxa, such as fish, amphibians, reptiles, and birds has entered the literature in the last two decades in the form of several excellent texts. Additional texts covering the pathology of wild animals in specific geographical areas, such as Europe and Australia, have augmented the recent literature. However, none of these has attempted to provide a single comprehensive reference, including infectious and noninfectious diseases, as is presented in this new book on the pathology of wild animals free-ranging and in managed care.

Lessons learned from the study of pathology of free-living animals can help us manage those in captivity, and vice versa. In fact, it could be argued that the distinction between free and captive with regard to wild animals is no longer entirely valid. There is little wild left anymore. Even free-living wild animals are constrained and managed by regulatory or physical boundaries imposed by human activity and anthropogenic environmental damage. We are living in the Anthropocene Epoch in which human induced changes are altering the geography and climate of our planet leading to large scale extirpation and extinction of numerous species. It is thus, more important than ever that we be concerned with the diseases that affect the health of individual wild animals and populations so that they may survive to be a part of healthy ecosystems and to inspire future generations.

It is anticipated that this book will provide a base for future studies on wild animal pathology. There is still much work to be done by veterinary pathologists in descriptive pathology and, even more so, by pathologists, clinicians, and biologists in scientific enquiry and hypothesis driven pathogenesis research to help us better manage and care for the fascinating panoply of animals found in the wild and in zoos and aquariums. This book is an invaluable reference for that endeavor.

Linda J. Lowenstine, DVM, PhD, Diplomate ACVP

Davis, CA.

Preface

It is indeed a fool’s errand to attempt the creation of a text describing the pathology of terrestrial and aquatic wildlife and zoo animals that occur throughout the world. That was our goal when we conceived this book. However, gaps in the documentation, descriptions of disease, and for many (arguably most) species on the planet the absence of a basic understanding of ecology and biology limited this lofty endeavor. The final product is a much humbler, basic introduction to the more commonly described and, in some cases, novel or emerging diseases of nondomestic species. We hope this text will serve as a reference for the variety of professionals and students working on wildlife and zoo animals, and as a resource for further scientific inquiries and descriptions.

Throughout the book, there is information on unique anatomy, epidemiology, and disease pathogenesis that will be applicable to biologists, researchers, clinicians, and veterinary pathologists who are already versed in diseases of domestic species. The text is organized using a set of introductory chapters that cover issues and themes applicable to a wide variety of species. For simplicity and ease of reference, the remaining chapters are organized by taxonomic groups. Each of these chapters begins with an introduction, including basic biology/ecology of the group along with unique gross and histological features that differ from the closest domestic (or farmed) animal paradigm. For some of the more unusual taxa, additional, detailed normal anatomical and histological features are described and illustrated in the supplemental online materials. Descriptions of gross necropsy lesions and keys to histologic and disease diagnosis for noninfectious and infectious diseases follow. For histologic descriptions, we made the assumption that the reader has a basic understanding of histologic terms and concepts, and unless otherwise stated in the figure legends, histologic sections were stained with hematoxylin and eosin. Descriptions of diseases that impact more than one group are thoroughly described with the taxa in which the disease is classically described (the canonical species/taxa chapter) with reference to it and species-specific differences highlighted in other chapters. For example, the characteristic gross and histologic lesions and pathogenesis of canine distemper virus (CDV) are described in the Canidae, Ursidae and Ailuridae chapter, with species-specific discussions appearing in chapters such as Felidae and Procyonidae, Viverridae, Hyenidae, Herpestidae, Eupleridae, and Prionodontidae. While we have tried to limit overlap, there is some intentional redundancy. As with unique features, information and data related to taxonomic lists, Latin and common names, species within a given taxa, histologic normal variations, and a number of diseases that are important but could not be accommodated in the chapter can be found in the supplemental online materials. We’re also extremely excited to host a dynamic set of more than 200 whole scanned slides on a companion website as a compliment to the still images in the book. The scanned slides provide valuable histologic material for those interested in furthering their knowledge on specific species and topics. Along with chapter authors, we endeavored to include reference lists containing as much of the primary literature and important seminal work as could be accommodated. As the editors of this book, we regret that we were not able to provide more extensive literature reviews for each chapter due to our lack of access (especially for publications in the non-English literature) and space limitations. Each chapter was written in good faith and with the goal of accurately describing diseases in wildlife and zoo animals. Any errors of interpretation and omission of important information, data, and publications lie solely with us, the editors. For these errors and omissions, we humbly submit our apologies and appreciate your constructive feedback and input.

Somebody needs to write a textbook is a phrase that we heard and said ourselves for a long time, not thinking that somebody would be us. We could never have accomplished this without our many mentors and colleagues who contributed as chapter authors. We are forever indebted to them for sharing their expertise and knowledge in these pages. From Academic Press, we would like to thank Pat Gonzalez for her patience in shepherding this book from conception to reality. We would also like to thank Laura Colantoni and Kathleen Reid for helping us navigate the publishing process at Elsevier, our acquiring editors Kristi Gomez and Anna Valutkevich, publisher Andre Wolff, Sreejith Viswanathan in production, and Melissa Dillon and Tyson Sturgeon for helping to enhance the online material. We would like to thank our coworkers who were not only recruited as authors but also covered service when editorial deadlines loomed. We would like to thank Lexi Mena for her outstanding assistance and contributions to image organization, editing, and formatting; and Alfred Ngbokoli and Kerry Prendergast and Debra Levinson for providing excellent histology and library services. Our thanks are also extended to our bosses Rick Fredrickson, Paul Calle, Brad Andrews, and Chris Dold for their support during the entire process. They were able to envision the final result and without them, it would not have been realized.

All books are harder to pull together and take longer than expected. None have been more significantly impacted during this process than our families. We recognize and truly appreciate the sacrifices, lost weekends, missed vacations, early mornings, late nights, and much more that you have endured in helping us achieve this monumental goal, and want you to know how very much your love and support through this process has meant to us. We would, from the very bottom of our hearts, like to specifically recognize and thank Dan, Abigail and Emmet Winter, Tylis Chang, and Marc Kratzschmar for your emotional and spiritual support; for doing a lot of housework, farm work, cooking many dinners, and providing wine; and for basically being your amazing selves. We absolutely could not have done this without each and every one of you!

Karen A. Terio

Denise McAloose

Judy St. Leger

Chapter 1

Wildlife Necropsy

Denise McAloose*

Kathleen M. Colegrove**

Alisa L. Newton*

*    Wildlife Conservation Society, Bronx, NY, United States

**    University of Illinois, Zoological Pathology Program, Brookfield, IL, United States

Abstract

A necropsy examination is one of our most important and, in some settings, our only opportunity to collect valuable diagnostic and archival samples from a wild animal. This chapter provides a practical approach to necropsy examination of vertebrate and invertebrate species, which includes sample collection for histology and ancillary diagnostic testing, and sample and data management and sharing.

Keywords

ancillary diagnostic testing

necropsy

postmortem examination

sample collection

Introduction

From the Greek for pathos (suffering) and logia (study of), pathology is the study of diseases, their cause or etiology, pathogenesis, and the structural and functional changes (clinical signs or symptoms), they produce. Clinical (or laboratory medicine; see Chapter 4) and anatomic pathology are the two main branches of this specialized medical field. In veterinary medicine, a postmortem or necropsy examination on a dead animal provides a significant opportunity to understand normal anatomy, biology, and the causes and various manifestations of disease. The aim of this chapter is to provide practical information for prosectors and pathologists performing necropsy examinations and disease investigations on nontraditional species both in individual animals and in outbreak situations.

Thorough postmortem or necropsy examinations capture all information relevant to the death of an animal or group of animals. When considered narrowly, results provide information about a single animal. More broadly and depending on the context, the compilation of historical and animal-related data forms the basis of understanding disease and the impacts of pathogens at the individual, population, species, and ecosystem level. These data may be locally, regionally, or internationally relevant for wildlife, zoo, agriculture, and companion, animals and for human public health. Necropsies are performed for many reasons. These include (but are not limited to) health and disease surveillance programs; characterization of normal and abnormal gross and morphologic anatomic features; establishing baseline health parameters and normal reference ranges; identification of the cause/s of morbidity and mortality in individual animals, groups, or populations; contribution of data and biomaterials to short and long-term research; determination of the effectiveness of medical or husbandry interventions or mitigation activities; collection of forensic information necessary in legal proceedings and prosecution (see Chapter 2), and in teaching and training. In conservation efforts, necropsy data is also important in rescue, recovery, reintroduction, and translocation programs. For example, to provide information about disease presence/absence in assurance colonies or relocation animals as well as in endemic populations and sympatric species to prevent unintended disease transmission. Results can additionally be used in the establishment of protected areas or to influence policy decisions (e.g., habitat use and resource extraction).

Protocols and procedures for laboratory or field based necropsies for a number of terrestrial or aquatic, domestic and nondomestic species are available from a variety of sources. These include governmental, nongovernmental, university or zoo based biology, veterinary medicine, and conservation organizations. Historically and to date, most were only available in text books or printed manuals. Related information can now also be accessed online with some of it in the form of instructional videos. A few examples of online protocols are those available through The World, European, or American Association of Zoos and Aquariums (WAZA, EAZA, and AZA, respectively), Taxon Advisory Group (TAG) or Species Survival Plan® (SSP) Programs, and the International Union for the Conservation of Nature (IUCN), though there are many others (Munson, 2000; Woodford, 2000). Some provide general instructions while others are taxon, genus, or species based. For some species, for example, invertebrates, there is value in reviewing basic zoology and biology references in addition to contacting colleagues with species specific expertise to discuss unique anatomic features and common normal and abnormal findings. Some of the latter can be found in the taxon based chapters and Supplemental Materials of this book.

Necropsy basics

Personal safety, the safety of the team, biosecurity, and communication are of paramount importance during any necropsy examination regardless of scale or scope. The complexity and coordination of these activities will differ between an individual animal death and disease outbreak or mass mortality event.

Personal protective equipment (PPE) includes gloves, surgical masks (or respirators), aprons, boots, and dedicated clothing (e.g., coveralls and surgical scrubs) (Box 1.1). Some combination of PPE should be worn during all necropsy procedures, cleanup, and carcass disposal. The type of PPE needed is informed by a risk assessment that takes into account the common diseases in a particular and sympatric species or in a collection or region. In some situations, prophylactic vaccinations are also needed to safely perform necropsy procedures. In addition to basic PPE, eye protection should be worn during venomous animal necropsies or to protect against splash risk. Additional attention should be paid to proper PPE any time equipment that can aerosolize tissues or pathogens (e.g., high pressure hose, drills, and saws) is used. Properly fitted N95 respirators or powered air purifying respirator (PAPR) should be worn when performing necropsy examinations on animals that pose a risk as carriers of zoonotic diseases and those that can be transmitted via aerosol routes (e.g., SARS in civets, tuberculosis in great apes, and elephants). Rabies vaccination or proof of protective titer should be standard if working with carnivores and other mammals. A necropsy should not be performed in cases where the risk to human health exceeds what one can be protected against with the available PPE (e.g., suspected hemorrhagic fever/ebola, and anthrax) or vaccination status. Veterinarians and human health professionals are typically responsible for making decisions about PPE and necessary vaccinations based on assessments that rely on an understanding of specific and relative risk, knowledge of infectious diseases, institutional standard operating protocols, and best practice protocols [e.g., United States Centers for Disease Control (USCDC), US Department of Agriculture, Office International des Epizooties (OIE), (PREDICT, 2016a; Richmond et al., 2003)].

Box 1.1

   Necropsy Examination Personal Protective Equipment (PPE), Supplies, Equipment, and Tools

This is a general guide that can be tailored to meet the specific situational needs of a particular necropsy examination/s.

Documentation

• Forms: Necropsy protocol and form, morphometrics data sheet, tissue sample checklist, human interaction form, notebook/paper [regular and waterproof (e.g., Rite in the Rain®)]

• Labeling: Tape, laundry tags with metal clips, pencils, waterproof marking pens, and pencils (to label samples that will be immersed in liquid fixatives), Tyvek®a

• Photodocumentation: Digital or film camera, batteries, memory cards, labels (include in every image)

Safety and Basic PPE

• Clothing: Gloves, mask (surgical, N95, masks with integrated face shield, respirator or powered air purifying respirator [PAPR]), eye protection (goggles, face shield), surgical scrubs, laboratory coat, coveralls, Tyvek®, or similar disposable fluid resistant coveralls, aprons, boots, gloves, caps (head cover)

• Disinfection: Sponges, dish soap, scrub brushes, disinfectant, bleach, alcohol (70% ethyl alcohol)

• Other: First aid kit, communication link (e.g., satellite phone), SDS (safety data sheets)/MSDS (material safety data sheets)

Tools

• Cutting: Scalpel blades, scalpel handles, knives (e.g., 6 or 8 blade), knife sharpenersb, scissors (small and large), bone shears, handsaws (e.g., hack saw, reciprocating saw), axe/hatchet, mallet/hammer, cutting boards, rongeurs, loppers (hedge clippers), chisel/wedge (e.g., T-shaped)

• Tissue handling: Forceps, meat hooks

• Containers: Rigid leak-proof wide-mouth spill-proof screw top containers (various sizes), zip-top plastic bags and Whirl-paks® (various sizes), serum tubes for fluid, blood and urine collection, aluminum foil, Teflon® bags, cryovials, sterile vials/containers/bags, sterile needles and syringes (various sizes), trochar (various sizes)

• Morphometrics (metric): Ruler, calipers, tape measure, scales

• Sterilization: Sterile instruments, matches or propane torch and searing blade/spatula, isopropyl alcohol for flaming instruments

• Culture: Sterile swabs, urine cups, and bags, culture transport media/tubes (for bacteria, virus)

• Tissue fixation: 10% neutral buffered formalin, 4% glutaraldehyde, or other EM fixative in small screw-top vials, isopropyl alcohol (for ecto and endoparasites, cytologic preparations, etc.)

• For genetic/molecular diagnostics (aliquot into small screw-top vials): 20% DMSO/saturated saline solution (genetics), RNA-later®, or TRIzol® (molecular diagnostics)

• Lighting: Head lamp, flashlight, batteries, light bulbs generator with extra bulbs, fuel

• Cold chain: Ice chest, ice packs, refrigerator, freezer (−20˚C, −80˚C, dry ice, liquid nitrogen), absorbent packing materials

• Laboratory equipment: Microscope [for field settings: field adapted (mirror as a light source), car battery adapted power source, generator], centrifuge

• Other: Ropes/straps/chains, string/suture, parafilm, glass slides and slide boxes, water supply/source (for cleaning/clean-up), plastic tarps, plastic tape/ropes to cordon off necropsy site, garbage bags, biohazard bags, disinfectant, bleach, sponges/scrub brushes, paper towels, portable generator (for electric powered saws, refrigerators, etc.)

Additional equipment for small carcasses (<5 g)

• Magnification: Dissecting microscope, magnification headband or surgical loupe

• Cutting: Microdissection forceps, scissors

Additional equipment for megavertebrate (whale, elephant, etc.)

• Cutting: Flensing knives, chain saw, large hammer/mallet and chisel, appropriate sharpener/s, shovel

• Tissue handling: Large meat hooks, gaff hook

• Morphometrics (metric): 20 m long (min)

• Mobility (for moving carcass, appendages, etc.): Hoist/crane with large capacity mounted scale

• Containers: Large sealable containers (e.g., vials to garbage cans).

• Other: Thick rope/chain (min of 20 m long), block and tackle

a When labeled with permanent marker or pencil may be placed in liquid (e.g., formalin) for identification purposes.

b For standard knives, recommend hand held sharpeners not sharpening stones since the use of stones requires significant expertise and inexperienced operators may dull rather than sharpen cutting instruments (e.g., knives, scissors).

Adapted from www.seadocsociety.org/wp-content/uploads/Orca-necropsy-protocol-FINAL-May-15-2014.pdf (Munson, 2000; Woodford, 2000).

It is also important, especially in field situations, to evaluate local environmental conditions to identify problems that pose safety risks, such as cold or rain, rising tides, or suboptimal animal position. In some situations, creative solutions or enlisting help from others (e.g., heavy machinery to move or position an extremely large carcass) will correct the situation and allow an examination to be safely performed. In others, selective or abbreviated necropsy procedures, or abandoning the necropsy altogether, may be the best and safest solution. Considering the safety of the media or the public and their pets is also important. Addressing this may require restricting access to the necropsy site with pylons, tape, or rope and providing restricted access signage or designated personnel for crowd control; local law or other wildlife enforcement agency presence may be necessary to protect the team and/or the public under certain circumstances.

Whether in a laboratory or the field, the best outcomes involve following standard protocols and having back-up plans. This is equally important when responding to a mass mortality event or performing a necropsy on an individual animal in a laboratory. You can’t plan for all possibilities. However, it’s important to work out the details of your situation-based needs and communicate the plan and contingency plans to those involved in the necropsy as well as to those providing logistical, technical, medical, and financial support.

In advance of a necropsy, it’s important to assign roles and responsibilities. Standard tasks include identification of a lead prosector/pathologist, and individuals or teams to: collect morphometric data; perform prosection; label and manage samples (diagnostic, research, archival); perform photodocumentation; manage data/data sheets (record, upload, and organize) and ensure fulfillment of all protocols; ship samples; perform cleanup, disinfection and carcass disposal; and manage communication within and between necropsy investigative team members and stakeholders, including the media. The least complicated situations are those involving a single animal and prosector in a controlled setting, like a necropsy suite or a laboratory. More complicated situations, for example, beach necropsies of whales, confiscations, or outbreak situations or mass mortality events, will involve a greater number of people from highly trained experts to unskilled volunteers, and a more formal and structured set of guidelines, protocols, reporting structure, and chain of command (see Mass Mortalities and Outbreaks below).

Just as there are laws and regulations that govern jurisdiction and handling of wildlife and zoo species, permissions to perform postmortem examinations and collect and distribute tissue samples are also required; the two are not always the same, even in zoo settings. It is therefore important to understand under whose jurisdiction (e.g., native peoples, state or federal wildlife agencies) a species is managed and what permissions are needed to perform necropsy examinations. In addition, the conditions of a permit may require the holder to provide reports or data to the permitting organization. It is therefore important not only to obtain permission to perform necropsies, but also to understand and comply with the restrictions and requirements associated with that permission.

If at any point before, during, or after a necropsy examination or mortality investigation, there is suspicion or confirmation of a locally, regionally, or internationally reportable/notifiable disease (e.g., USDA, CDC, or OIE reportable disease) or if criminal activities are suspected, appropriate authorities must be immediately contacted and activities coordinated. In legal or forensics cases, collection of data and samples using appropriate chain of custody and documentation (see Chapter 2) is also necessary. Structured reporting systems exist in every country. It is important to be familiar with those systems that apply in the jurisdiction in which you work. Direct contact with regulatory agencies or online information will provide the most up to date information about those diseases that are listed and reportable (www.oie.int/animal-health-in-the-world/oie-listed-diseases-2017/; https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/monitoring-and-surveillance/sa_disease_reporting/ct_disease_list; https://www.agriculture.ny.gov/AI/disease_rep.html).

Necropsy examination

Preparation

Before a postmortem examination begins, it’s worth remembering that you are the most valuable asset in the process. All conclusions drawn in a mortality investigation rest on the information and samples you and your team collect. Best intentions and memory are no substitution for the collection of written and photographic documentation. Also, since it is uncommon to retain an entire carcass for sampling after death, the postmortem examination is your first and perhaps only opportunity to collect circumstantial evidence, diagnostic samples, and to permanently record the information from which relevant death factors and baseline information are established. Each person involved in the necropsy must take personal responsibility for their actions. Essential in all of this is focus and the engagement of your brain and critical senses: sight, smell, touch, and hearing. Outcomes are as good as the data you collect and the focus you bring to the tasks. and what you do during and after the examination will make the impossible, possible.

The tools needed to perform a necropsy examination will vary based on the number, size, and shape of the carcass/es (e.g., dissecting scope for very small animals, hoists, cranes, and power tools for large animals). Box 1.1 contains a general list of necropsy supplies, tools, and equipment that can be modified to appropriately address the species and circumstances at hand.

The Essentials

All mortality investigations, regardless of whether they involve a single animal or a large mortality event, share a set of consistent elements: an iterative diagnostic plan (Fig. 1.1) that applies evidence against a set of case definitions (Box 1.2) and differential diagnoses to establish a cause of death. Central in this process is the necropsy examination. Providing specific necropsy protocols for all taxa and for every situation is beyond the scope of this chapter. However, all necropsy procedures follow a similar, basic format.

Figure 1.1   Mortality investigation flow chart.

All mortality investigations include a consistent set of standardized activities. These include collection of information related to the circumstances of an animal’s death, gross necropsy and sample collection for ancillary diagnostic testing, and establishing a set of differential diagnoses and case definitions against which test results are applied in order to arrive at final diagnoses and conclusions. Integral in this process is collection of information and documentation of all findings, and clear and consistent communication with all relevant stakeholders.

Box 1.2

   Case Definition

A case definition is a mechanism for categorizing animals as having or not having a disease. It is based on a clearly stated set of criteria that includes history (e.g., epidemiological, temporospatial, medical, husbandry), signalment (e.g., species, age, sex), clinical signs, and necropsy, histology, or other diagnostic test results. Fulfillment of the criteria determines whether an animal is considered a case. In some situations, multiple case definitions may be used.

A case definition should contain criteria for inclusion as well as criteria for exclusion. For example, if the case definition requires that affected animals are neonates, an animal in another age class (e.g., juvenile, subadult, adult) would be excluded and not classified as a case. Sometimes the absence of critical information is sufficient for exclusion of a case from a series. For example, if sex is important, then absence of documentation of the sex of the carcass can result in exclusion of an individual case from a series even if all other information is of high value.

Cases can be classified using the following criteria:

Confirmed: Fulfills all criteria for inclusion including positive laboratory results

Probable: Fulfills all or most of the criteria for inclusion but lacks laboratory confirmation

Possible: Has some but not all criteria for inclusion but lacks laboratory confirmation

Negative: Does not fulfill the criteria for inclusion and lacks laboratory confirmation

Collection and Documentation of Relevant Historical Information

In addition to information that will be collected from the carcass, it is also important to collect and record relevant clinical and death-related information. This includes basic (e.g., species, breed, gender, age/age group, birth and death dates) and medical information about the subject animal, contact animals, and other sympatric contact species (wildlife/vermin, humans, pets, livestock, etc.). A complete data set also includes health and disease-related epidemiological (number of deaths, spatial/temporal events, etc.) and environmental information. Carefully examine the area and collect information about the location in which an animal was found dead (e.g., GPS location, weather, environmental conditions, changes in husbandry practices, evidence of a struggle) to record those things that may be relevant to the circumstances of an animal’s death. For example, weather patterns or environmental sampling of soil/sediment or water in aquatic settings may be important in die-offs of multiple or large numbers of animals and/or when there are deaths across species or taxa. These and other types of information may be of importance as forensic evidence in a legal case (see Chapter 2).

External and Internal Examination

A good rule of thumb before performing a necropsy procedure is to familiarize yourself with the normal anatomy and natural history of the species on which you will be working (e.g., normal diet, fecal consistency, color patterns, sexual dimorphism, common and zoonotic diseases). This is particularly important when performing a necropsy on a species with which you are not familiar and it will inform activities related to personal protective equipment and risk. Taxa specific chapters within this text, other reference textbooks, online resources, and colleagues with species specific knowledge are valuable in this regard. It is also worth remembering that while every taxa and species has unique features, familiarity with one often serves as a helpful reference for another. For example, familiarity with the anatomy of well described and studied domestic or farmed species is directly applicable to the anatomy of most nondomestic mammals, fish, and birds. Invertebrates provide a greater challenge. This is an incredibly diverse group that includes more than 99% of all animal species on the planet. Over the past decade, there has been increased interest in this group and expertise and resources are increasing. Recommendations below and in the invertebrate chapter in this book (see Chapter 41) provide basic anatomy and an approach to invertebrate necropsy.

Even in this age of digital photography, written descriptions of visual observations (animal, physical environment, etc.) remain a critically important component of a necropsy examination. Two important general rules of thumb during a necropsy are to describe what you see and limit descriptions to factual observations rather than interpretations or diagnoses. For this, it is worth remembering that you describe all sorts of things every day and the language you use to describe what you see during a necropsy examination is no different than the language you use during every day conversations. Basic necropsy descriptions include information about: location, number, color, size, shape, distribution (e.g., focal, regionally extensive, diffuse), consistency and texture (soft, firm, hard), and odor. For example, dozens of pinpoint to 5 mm in diameter, soft, light tan nodules that contain thick, white material on cut section are present throughout the liver. Weights of organs are important to establish normal reference intervals and for organs thought to be too large or too small. Size measurements should be taken in three dimensions (length × width × height/thickness). Remember, a description should include those details that allow someone who did not witness the necropsy to create an accurate mental image of what you saw. Also, remember that a description is not an interpretation or a diagnosis. Those are best left to the pathologist to formulate based on your description and other documentation (e.g., photographs). For example, differentials based on the earlier description include abscesses, hyperplasia, or cancer. The description is the same regardless of the pathologist’s final diagnosis, which is based on a combination of the gross description as well as histology and ancillary diagnostics. For photodocumentation, always include a size reference (a ruler is best) and animal related information (minimum: identification number and date). Rulers and identification tags should be placed next to and should not obscure the lesion. All information from the necropsy and collected samples should be entered into logs and secure databases developed for this purpose. This is important for a whole host of reasons including rapid data retrieval and collation during mortality events, and data and sample sharing for immediate and future research projects. Information should be as detailed as possible to ensure that important information is not forgotten or lost.

Under ideal conditions, a necropsy is performed and samples are collected from freshly dead animals. If necropsy will be delayed, carcasses should be refrigerated or kept cool on ice for several hours to a few days; freezing should be an option of last resort as the freeze/ thaw cycle will alter the color of many tissues, kill many pathogens (e.g., bacteria and fungi), and ice crystal formation will significantly limit the value of histology. However, under some circumstances, for example, when a necropsy cannot be safely, thoroughly, or adequately performed in a timely manner, a necropsy can be performed on previously frozen carcasses though the results will not be optimal.

All necropsy examinations should by systematic and follow standardized procedures and protocols. This ensures that the carcass is thoroughly examined, standardized information for generating baseline and documenting abnormalities is captured, and a complete set (or sets) of data and samples are collected for baseline, diagnostic, archival, and research purposes. There are several common approaches to a necropsy examination. One is not necessarily better than another and your preferred method may vary depending on the species and specific circumstances. Regardless of your method, of paramount importance is that you work safely and have a consistent and systematic strategy for organ and organ system examination. This is especially important when working in large teams or in complicated situations (e.g., mass mortality event). It is also important to work cleanly to maximize visibility and minimize contamination of the worksite and exposure risks. Where applicable, standardized necropsy procedures and/or sampling/research associated requests that are required by government agencies should also be followed. When practical, fulfilling biomaterials requests allows valuable, additional information to be generated.

The external examination is an assessment of the tissues outside of the main body cavities. The exam begins with direct measurement or estimation of carcass weight, collection of morphometric data, photodocumentation, and assessment of state of decomposition. Every carcass, regardless of its state of decomposition, can provide valuable scientific data. In many field situations, access to fresh carcasses is the exception rather than the rule. Environmental conditions (e.g., high humidity, high temperatures or dense foliage in natural or recreated settings) and interval from death to discovery all affect the degree of decomposition. Tissue degradation varies by tissue type and affects the reliability of diagnostic test results. It is therefore important to document the degree of carcass decomposition, otherwise known as carcass condition, to contextualize observations and diagnostic test results. This is a subjective assessment and a relatively common scheme categorizes postmortem carcass condition as excellent (freshly dead) to mildly autolyzed, moderately autolyzed, severely autolyzed, or skeletonized/desiccated/mummified remains. In the marine mammal community, several protocols employ a similar carcass condition coding system (Box 1.3). Assessment of carcass condition is particularly useful in low resource or time restricted settings to identify those carcasses that will be the most diagnostically valuable and to direct sampling and testing.

Box 1.3

   Carcass Condition Codes

Code 1: Live animal.

Code 2: Fresh/mild decomposition. The body is intact. The skin is firm with little drying or wrinkling of the skin, eyes, and mucous membranes. The eyes are clear. The abdomen/gastrointestinal tract is not distended with gas. The tongue, penis, anus do not protrude.

Code 3: Moderate decomposition. Intact carcass but there is distention (bloating) of the abdomen due to gas accumulation. The tongue, penis, rectum may be protruding, especially in large animals. The skin is dry and the hair may epilate easily with manual handling. The organs are discolored but are largely identifiable.

Code 4: Advanced/severe decomposition. Intact, collapsed carcass. The hair and skin sloughs easily with manual handling. Some internal organs are intact and identifiable but others (e.g., gastrointestinal tract) are extremely friable or liquefied.

Code 5: Mummified or skeletal remains. Often all that remains are portions of dried skin, hair, and/or skeletal muscle over bones.

This scheme primarily describes assessment of vertebrates. A similar scheme can be applied with some modifications to invertebrates. Carcasses in condition code 2 are the most valuable for sample collection for histology and ancillary diagnostic testing. Many tests can also be run on samples collected from code 3 and 4 carcasses but caution should be used when interpreting results. A limited number of diagnostic tests, for example, skeletal morphometrics and some molecular diagnostic tests, can be run on code 5 carcasses; many caveats apply.

Adapted from: Geraci, J., Lounsbury, V. (Eds.), 2005. Marine mammals ashore: a field guide for strandings, second ed., National Aquarium of Baltimore, Baltimore, MD.

In vertebrates (see later for invertebrates), a systematic external examination with the animal in lateral or ventral recumbency includes evaluation of normal and abnormal findings in: the structures of the head (e.g., eyes, ears, nares, bill, beak, teeth, cere, gills, tonsil); the outer covering of the body (e.g., skin, hair, feathers, scales, claws, hooves); the subcutis, glands (e.g., periorbital, salivary, scent, salt, perianal, uropygial); external reproductive system tissues; the musculoskeletal system (including assessing muscle mass and one to multiple joints and bone marrow); and the peripheral nervous system. It also includes objective (measured) and/or subjective assessment of fat stores (e.g., mesenteric, pericardial, subcuticular, blubber). In some cases, skeletal preps (e.g., warm water maceration) and radiographs or other imaging modalities [e.g., radiography, computed tomography (CT), magnetic resonance imaging (MRI)] may expose or aid in documenting lesions. Written and photographic documentation of all notable normal and abnormal findings should be recorded and representative sample/s from all tissues and all lesions should be collected and placed into 10% neutral buffered formalin (NBF) for histology or collected as appropriate for ancillary diagnostic testing (Box 1.4 and Supplemental Materials for general forms and tissue checklists for vertebrates and invertebrates).

Box 1.4

   Examples of Gross Anatomy and Sample Collection Checklists

a,b

Vertebrate: Carnivore

Invertebrate: Echinoderm

a See the Supplemental Materials for additional information on invertebrate necropsy and gross anatomy and histology tissue checklists for: arthropods, cnidarians, echinoderms, bivalve and gastropod molluscs, and cephalopod molluscs.

b Note: These are general lists that should be modified to reflect the anatomy of the species being necropsied and any additional specific needs (e.g., research requests, specific pathogen screening). For example, collection of tissues from ruminants should include sections from the forestomachs (rumen, reticulum, omasum) and abomasum; fish would include gill and anterior kidney, etc. Collect one to multiple samples of normal and abnormal tissue and junctions between normal and abnormal. For bilateral organs (e.g., kidneys, adrenal glands), samples should be collected from each. Multiple parallel cuts should be made in solid organs to assess the paranchyma prior to sampling. Tubular organs should be opened along their long axis and examined prior to sampling. Tissues samples for histology should not be greater than 0.5 cm thick and should be placed in 10% neutral buffered formalin (NBF) for fixation (1 part tissue:10 parts NBF). In some animals, [small animals <~5 g, those that have fragile tissues (e.g., arthropods)], perfusion of the body cavity/ies or fixation whole with subsequent postfixation dissection may be preferable to gross dissection of the fresh animal.

The internal examination begins by making a ventral midline incision from the symphysis of the mandible to the inguinal region and reflection of the skin and limbs. This is followed by opening (1) the abdomen or coelom by removing the abdominal/coelomic wall distal to the xiphoid and ribs, (2) the thorax by cutting the diaphragm (when present), checking for negative pressure in the thorax, and removing the ribs unilaterally or bilaterally (depending on position), and (3) the pericardial sac to expose the heart (some modifications may be necessary based on species specific anatomy). It is standard practice to open these cavities and assess the general body condition of the carcass (e.g., assessment of cavitary fat stores, fat body size, and size of the liver) and examine all organs and organ systems in situ prior to tissue removal or sampling. As for external examinations, knowledge of domestic animal anatomy is directly applicable and serves as a guide for identification of tissues and organs in nondomestic species. It is standard practice when opening these cavities to immediately collect sterile samples (e.g., heart blood, abscesses, cavitary fluid, fungal plaques) for ancillary diagnostics, archiving, and to perform in situ photodocumentation before handling the organs. If care is taken to open the cavities with minimal handling, the risk of contaminating diagnostic samples is minimized and collection of tissues for archival purposes is more easily managed.

Three common approaches for performing detailed tissue dissection and sample collection after in situ examination are (1) removal of tissues in the thoracic cavity/cranial coelom and the abdominal cavity/caudal coelom en bloc, (2) removal of tissues by organ or organ system, or (3) a combination of the two. Preference as to which approach is taken, whether some modification of these is chosen, and the order of the examination is up to the prosector. Important considerations include the species, situational needs, known or suspected diseases, and general rates of autolysis (e.g., removal, dissection, and sampling of gastrointestinal tract tissues might occur before thoracic cavity tissues to minimize autolysis in diagnostically important tissues). Regardless of the approach, a thorough, systematic examination is essential. As for the external examination, written and photographic documentation of all notable normal and abnormal findings should be recorded and representative sample/s from all tissues and all lesions should be collected and placed into 10% NBF for histology (Box 1.5) or collected as appropriate for ancillary diagnostic testing and archiving. See Box 1.4, Table 1.1, and the Supplemental Materials for sample collection, storage and handling recommendations and general forms and tissue checklists.

Box 1.5

   Preservation Solutions

(Munson, 2000; Woodford, 2000)

Field preparation of 10% neutral buffered formalin (1 L)

• 100 mL commercial formalin (38%–40% formaldehyde)

• 900 mL distilled water

• 4 g sodium phosphate monobasic

• 6.5 g sodium phosphate dibasic (anhydrous)

1. Pre-weigh the sodium phosphates and place in vials in units suitable to the volume to be mixed (1 vial for each 1 L of buffered formalin to be prepared)

2. Fill a 1 L plastic bottle with 900 mL water, draw a line to mark the level; add 100 mL more and mark the 1 L line; empty the bottle for transport to the field

3. In the field, add 900 mL water to the bottle; add ¼ of the sodium phosphate powder and shake to dissolve; repeat until all powders is dissolved

4. Fill bottle to 1 L line with commercial formalin

Sterile buffered 50% glycerin

a

• Buffer

• Mix 21 g citric acid with 1 L distilled water

• Mix 28.4 g anhydrous sodium phosphate with 1 L distilled water

• Add 9.15 mL citric acid solution (A) to 90.85 mL anhydrous sodium phosphate solution (B)

1. Mix 100 mL buffer with 100 mL glycerin

2. Aliquot into smaller, leak-proof containers and sterilize for subsequent field use

a for transport of tissue samples for culture when refrigeration is not available

Carnivores

Depending on carcass size, carnivores are placed in dorsal or lateral recumbency. After completion of the external examination and sample collection, in situ examination, and collection of sterile samples and samples for ancillary diagnostics or archiving, the internal organs are removed and more thoroughly examined.

In the thoracic cavity, examine and remove the pluck en bloc. The pluck includes the tongue, oropharynx, and esophagus; thyroid and parathyroid glands; larynx, trachea, lungs, thymus, thoracic lymph nodes, and the heart and pericardial sac. Prosectors may elect to remove and collect samples from the thyroid and parathyroid glands prior to removing the pluck as they are relatively small and easily overlooked or lost during a necropsy. Examine and collect samples of tongue, thyroid, and parathyroid glands, thymus (if present), and lymph nodes. Open the esophagus along its length noting mucosal changes (e.g., discoloration, erosion, ulcer, and proliferation) and intraluminal content. Open the trachea along its length and the bronchi and major vessels into the lungs noting mucosal changes and any abnormal intraluminal content. Palpate the lungs, noting their