Wildlife Necropsy Primer

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Wildlife Necropsy Primer DENISE MCALOOSE

Introduction Thorough postmortem or necropsy examinations capture all of the information that is relevant to the death of an animal or group of animals. When considered narrowly, the results provide information about an individual. More broadly and depending on the context, the compilation of these data forms the basis of understanding disease and the impacts of pathogens at the individual and species, population, and ecosystem levels. These data may be locally, regionally, or internationally relevant for wildlife, zoo, agriculture, and companion animals and for human public health. A necropsy may be performed for many reasons, including (not limited to) 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 to short- and long-term health and disease surveillance and monitoring programs; establishment of the presence and significance of pathogens and disease in individuals, groups/populations, and ecosystems; determination of the effectiveness of medical or husbandry interventions or mitigation activities; collection of forensic information necessary in legal proceedings and prosecution; and teaching and training. In conservation efforts, necropsy data may be important in recovery, reintroduction, and translocation programs (e.g., to understand disease presence/absence in assurance colonies or relocation animals and endemic populations to prevent unintended disease transmission). Results may also be used in establishment of protected areas or to influence policy decisions (e.g., habitat use, resource extraction). Protocols and procedures for laboratory- or field-based necropsies for many terrestrial and aquatic domestic and nondomestic species are available through a number 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 textbooks or printed manuals. Much information can now also be accessed online, sometimes in the form of instructional videos. A few examples of online protocols are those available through the World, European, 194

or Association of Zoos and Aquariums (WAZA, EAZA, AZA, respectively) Taxon Advisory Group (TAG) or Species Survival Plan Programs (SSP, EEP) and the International Union for Conservation of Nature (IUCN), although there are many others.1,2 Some provide general instructions, whereas others are taxon, genus, or species based. In some cases, access to online protocols may need to be requested. For others, for example necropsy of nontraditional species like 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 and emerging diseases.

Necropsy Basics Personal safety, the safety of the team (which may include experienced individuals and groups, as well as enthusiastic but inexperienced staff, colleagues, or volunteers), biosecurity, and related communication are of paramount importance during any necropsy examination, regardless of scale or scope. In addition, the complexity and coordination of activities will vary and differ between an individual animal death and disease outbreak or mass mortality event. Basic personal protective equipment (PPE) including gloves, surgical masks (respirators), aprons, boots, and dedicated clothing (e.g., coveralls, surgical scrubs) (Box 30.1) should be worn during all necropsy procedures, clean-up and carcass disposal, and any time equipment that may aerosolize tissues or pathogens is used (e.g., high-pressure hoses, drills, saws). In addition, an understanding of the common diseases in a particular and sympatric species and in a collection or region and risk assessment inform additional PPE and prophylactic vaccinations that are needed to safely perform necropsy procedures (e.g., eye protection for venomous animals or infectious disease splash risk, properly fitted N95 respirators for aerosol transmitted zoonotic pathogens such as tuberculosis, rabies vaccination if working with carnivores, bats, or other susceptible species). In cases in which the risk to human health goes beyond management with available PPE (e.g., suspected hemorrhagic fever, Ebola, anthrax) or vaccination status, a necropsy should not be performed. It is also important, especially in field situations, to evaluate the local environmental conditions to

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• BOX 30.1  Necropsy Examination Personal Protective Equipment (PPE), Supplies, Equipment, and Tools

Documentation • Forms: necropsy protocol and form, morphometrics data sheets, 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®* • Photodocumentation: digital or film camera, batteries, memory cards, labels (include in every image)

Safety and Basic PPE • Clothing: gloves, mask (e.g., surgical, N95, masks with integrated face shield), eye protection (goggles, face shield), surgical scrubs, lab coat, 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/MSDS

Tools • Cutting: scalpel blades, scalpel handles, knives (6- or 8-in blade), knife sharpeners†, scissors (small and large), bone shears, handsaws (e.g., hacksaw, reciprocating saw), axe/hatchet, wedges, mallet/hammer, cutting boards, rongeurs, loppers (hedge clippers), chisel/wedge (e.g., T shaped) • Tissue handling: forceps, meat hook • Containers: rigid leakproof wide-mouth spillproof screw-top containers (various sizes), zip-top plastic bags and Whirlpak® (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 in-date 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 ectoparasites and endoparasites, cytologic preparations, etc.) • For genetic/molecular diagnostics (aliquot into small screwtop vials): 20% DMSO/saturated saline solution (genetics), RNA-later® or TRIzol® (molecular diagnostics) • Lighting: head lamp, flashlight, batteries, light bulbs, generator with extra bulbs and 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, surgical loupe • Cutting: microdissection forceps, scissors

Additional Equipment for Megavertebrate (whale, elephant, etc.) • Cutting: flensing knives, large reciprocating saw, large hammer/mallet and chisel, appropriate sharpener(s), shovel • Tissue handling: large meat hooks, gaff hook • Morphometrics (metric): 20 m long (min) tape measure • 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

*When labeled with permanent marker or pencil, may be placed in liquid (e.g., formalin) for identification purposes. † For standard knives, recommend hand held sharpeners not sharpening stones because the use of stones requires significant expertise, and inexperienced operators may dull rather than sharpen cutting instruments (e.g., knives, scissors). This is a general guide that may be tailored to meet the specific situational needs of a particular necropsy examination(s). MSDS, Material safety data sheets; PPE, personal protective equipment; SDS, safety data sheets. Data from http://www.seadocsociety.org/wp-content/uploads/Orca-necropsy-protocol-FINAL-May-15-2014.pdf; Necropsy procedures for wild animals. In White L, Edwards A, editors: Conservation research in the African rain forests: a technical handbook, New York, 2000, Wildlife Conservation Society, pp 196–217; Woodford M, Keet D, Bengis R: A guide to post-mortem procedures and a review of pathological processes identified in the elephant. In Woodford M, editor. Post-mortem procedures for wildlife veterinarians and field biologists. Paris, France, 2000, Office International des Epizooties, Care for the Wild and the Veterinary Specialist Group/Species Survival Commission of the World Conservation Union (IUCN).

identify problems that pose safety risks such as cold or rain, rising tides, or suboptimal animal position; in some cases where safety cannot be ensured, a necropsy should be postponed or abandoned. Wildlife and veterinary pathologists and clinicians and human medical health professionals are typically responsible for making these decisions based on a risk assessment that relies on an understanding of specific and relative risk, knowledge about infectious diseases and circumstantial situations, institutional standard operating protocols, and best practice protocols. In addition, if at any point there is a suspicion or confirmation of a locally,

regionally, or internationally reportable/notifiable disease (e.g., USDA [US Department of Agriculture], CDC [Centers for Disease Control and Prevention], OIE [World Organization for Animal Health] reportable disease) or illegal activities (e.g., poaching, poisoning) are suspected, the scene should be secured and appropriate authorities contacted before proceeding. For reportable/notifiable diseases, direct contact with regulatory agencies or online information will provide the most up-to-date information about those diseases that are listed (see http://www.oie.int/ animal-health-in-the-world/oie-listed-diseases-2017/).

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In advance of a necropsy, it is important to have organized plans and 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, organize), and ensure fulfillment of all protocols (including sample collection for research requests), ship samples, perform clean-up/disinfection and carcass disposal, and manage communication within and between necropsy investigative team members and stakeholders, including the media. Having organized plans and assigned roles is particularly important in megavertebrate necropsies or mass mortality events, due to the size or number of animals, participants of varied skill levels, and numerous different agencies that may be involved.

Necropsy Examination Before a postmortem examination begins, it is worth remembering that you, whatever your role, 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 substitute for the collection of real-time verbal, written, and photographic documentation. Each person involved in the necropsy must take personal responsibility for his or her actions. Outcomes are as good as the data you collect and the focus you bring to the task, and what you do during and after the examination will make the impossible, possible. All mortality investigations, regardless of whether they involve a single animal or a large mortality event, contain a set of consistent elements: a systematic, iterative diagnostic plan that applies evidence against a set of case definitions and differential diagnoses to establish a cause of death (Fig. 30.1). 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 (described below).

Collection and Documentation of Relevant Historical Information In addition to information that will be collected from the carcass, it is quite important to collect and record relevant death-related information. This includes basic animal information (e.g., species, breed, gender, age/age group, birth and death dates) and clinical medical information about the subject animal, contact animals, and other sympatric species (including other wildlife/vermin, humans, domestic animals, livestock, etc.), environmental, and epidemiologic information. It is also important to record information about the location in which an animal was found dead, environmental conditions/enclosure information, husbandry changes, and to note other findings (e.g., evidence

of a struggle) that may be relevant to the circumstances of the animal’s death.

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 (e.g., what is the best approach to opening the body cavity of a chambered nautilus [Nautilus pompilius]) and will inform activities related to PPE and risk. Numerous texts and online resources, as well as colleagues with species-specific knowledge, are good places to start. It is also worth remembering that, although every taxon 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 becoming more widely available. All necropsy examinations should be systematic, follow standardized procedures and protocols, and include checklists for the collection of “standard” sets of tissues (Box 30.2). This ensures that a 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. When practical, fulfilling sampling/research-associated requests (e.g., biomaterials requests) allows valuable, additional information to be generated. Where applicable, standardized necropsy procedures and/or sampling/research-associated requests that are required by governmental agencies should also be followed. Even in this age of digital photography, written descriptions of visual observations (animal, physical environment, etc.) remains a critically important component of a necropsy examination. But that does not mean that they need to be overly complicated. Two general rules of thumb during a necropsy: describe what you see and limit descriptions to factual observations rather than interpretations or diagnoses (this occurs later). Keep in mind 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 everyday conversations. Basic necropsy descriptions include information about location (e.g., organ, body system) number (e.g., 1, 10, more than 20 but less than 100, multiple, many, myriad), color, size, shape, distribution (e.g., focal, regionally extensive, diffuse), consistency and texture (soft, firm, hard), and odor. For

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• Figure 30.1

  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.

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.” Measurements are useful for documenting lesions, including weights of 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 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 (in the above description, it is hoped that you imagined multiple hepatic abscesses). For photodocumentation, always include a size reference (e.g., small ruler) and animal-related information (minimum: ID number, date). 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 current 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 because the freeze-thaw cycle will alter the color of many tissues and kill many pathogens (e.g., bacteria, fungi), and ice crystal formation will significantly limit the value of histology. However, under some circumstances (e.g., when a necropsy cannot be safely, thoroughly,

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• BOX 30.2  Sample Collection Checklist Skin Umbilicus

Thyroid glands (neonates)

(both)

Parathyroid glands

Stomach

Adrenal glands

Small intestine

Kidney

(cardiac, fundic, pyloric)

Skeletal muscle

Trachea

Peripheral nerve

Esophagus

Large intestine

Heart

Liver

(e.g., sciatic)

Bone/bone marrow

(e.g., end of long bone, rib)

(free walls and septum with valves)

Salivary gland

Lung

Lymph nodes

(each lobe and a bronchus)

(e.g., popliteal, bronchial, mesenteric, ileocolic)

Thymus

Tongue

Diaphragm

(with and without pancreas)

(bisected longitudinally) (bisected longitudinally; include cortex, medulla, pelvis)

Ureters

Penis Ovaries Uterus Cervix Vagina

(with and without capsule)

Urinary bladder

Gallbladder

Urethra

Pancreas

Accessory glands (e.g., prostate)

(whole or cut longitudinally along midline)

Spleen

Testes/epididymis

Spinal cord

(with and without intestine) (with and without capsule)

(bisect longitudinally)

Tonsil Brain

Eye

*This is a general list 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) while samples from fish would include gill and anterior kidney. 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 the left and right sides. Multiple parallel cuts should be made in solid organs (e.g., liver) to assess the parenchyma prior to sampling. Tubular organs (e.g., intestine) should be opened along their long axis and examined prior to sampling. Tissue samples should not be greater than 0.5 cm thick and should be placed in 10% neutral buffered formalin (NBF) for fixation (1 part tissue to 10 parts NBF).

or adequately performed in a timely manner), a necropsy can be performed on previously frozen carcasses, although the results will not be optimal. The external examination is an assessment of the tissues outside the main body cavities. This 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, may 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. As the body decomposes, all tissues degrade. The speed at which this occurs and the reliability of diagnostic test results that may be obtained from different tissue types during this process vary. 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 use a similar carcass condition coding system.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 (e.g., those that are “freshest” or most intact) and to direct sampling and testing. In vertebrates a systematic external examination of the carcass 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), and skeletal muscles; external reproductive system tissues; the skeletal system (including one to multiple joints); and the peripheral nervous system. It also includes objective (measured) and/or subjective assessment of fat stores (e.g., subcuticular, blubber) and muscle mass and collection of bone marrow (typically femur or rib in nonavian and tibiotarsal bone in avian species). 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. An internal examination is an assessment of the organ systems and individual organs in the body cavities. There are a number of common approaches to an internal examination. One is not necessarily better than another, and your preferred method may vary depending on the species and specific circumstances. Regardless of the method, of paramount importance is that you work safely, have an organized plan, and have a systematic strategy for organ and organ system examination to ensure all tissues are examined, lesions documented, and samples collected. This is especially important when working in large teams, on large animals, or in complicated situations (e.g., a mass mortality event). It is also important to work cleanly to minimize, as much as possible, contamination of the worksite and exposure risks to personnel and other animals (e.g., scavengers).

CHAPTER 30  Wildlife Necropsy Primer

Species/Age-Specific Considerations There are a number of notable, normal features to be aware of when performing necropsies on neonates. In mammals and birds, these include skeletal muscles that will generally be lighter in color than in adults; soft, light tan/orange liver (due to hepatocellular glycogen stores); low amounts of subcuticular or cavitary fat; and a very soft brain even in very fresh carcasses. In term mammal neonates, the ductus arteriosus and foramen ovale may be probe patent (should be functionally closed) and heal within the first few days of birth, the gonads and adrenal glands may appear relatively large, and if the carcass was refrigerated after death, the lenses may become opaque white (rather than translucent). In birds an egg tooth and (occasionally prominent) pipping muscle are seen. Birds and reptiles will have an internalized yolk sac, which may occupy a majority of the caudal coelom. As in their domestic counterparts, important abnormal findings include the presence of congenital abnormalities or defects (e.g., cleft palate, extra or missing limbs or limb deformities, cardiac septal defects) and excessive numbers of squames, meconium, yolk, or other foreign material in the lungs on histology. They may also have incompletely internalized yolk sacs (suggestive of premature pipping or hatch complications) or subcutaneous edema (e.g., head, neck), which may reflect suboptimal incubation parameters. In eggs the content of the air cell (it is normally empty; cultures of accumulated fluid or membranes may be taken at this point), condition of the membranes (color, intact, pipped, etc.), and assessment for fertilization (e.g., presence of blood spot, embryonic membranes) and the developmental stage, and position of a developing fetus/ embryo should be assessed.6 Specific precautions should be taken when performing necropsies on venomous snakes, lizards, amphibians, fish, invertebrates, and mammals. Only those with experience and knowledge of the type, strength, and mechanism of actions of venom, location of production, and means of envenomation (e.g., rear fangs, spines, spurs, secretions) should handle venomous animals. Handling, PPE, and emergency protocols should be established before and followed during the necropsy. For example, venomous snakes should be placed in a completely secured, seethrough container for transport, information about the species and antivenin should be attached/submitted with the animal/container, and the head should be taped or otherwise secured in such a way that the mouth is not free to open. Guidelines for contacting emergency/first responders, an envenomation center, emergency hospital, zoological institution or others that maintain antivenin stocks, and an emergency hospital with expertise in managing envenomation patients should be included in these protocols. For venomous snakes, the recommended first step in the necropsy procedure after confirming the snake is dead by observation through the see-through container is to remove the head and place it into formalin. This accomplishes two tasks, deactivation of venom and tissue fixation. However, it is not known if formalin deactivates all venom proteins, and

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tissue fixation may be incomplete in deep tissues, especially in large heads, so continued caution is warranted whenever the head is handled. Cytologic examination of skin (and external mucus layer), fin tissue, and gill clips to identify ectoparasites, fungus, or other pathogens should be performed as a first step in a fish necropsy. Invertebrates present a challenge at necropsy, due to the myriad species and body types represented by this vast group of animals. Critical to gross and histologic evaluation, as with all species, is first reviewing basic textbooks and journal publications on anatomy, biology, and published diseases7–12 and, through this, generating a checklist of organs and organ systems for consistent gross and histologic evaluation. In some species, gross evaluation will significantly disrupt tissue architecture. This is particularly true of arthropods and bivalve mollusks. In those cases, limited external evaluation of the ectoskeleton (carapace) and soft tissues, and collection and examination of hemolymph and cytologic (wet mount) evaluation of gills and external abnormalities are recommended prior to fixing the entire animal for histologic evaluation. For very large animals, heavy equipment may assist in moving the carcass, limbs, and organs, as well as for carcass movement or burial. For very small animals (<5 g), the use of a dissecting microscope or head loupe is an incredibly helpful tool. In some cases the best option is to fix an entire animal. This may be achieved by either making a small incision in the body cavities to allow fixative to contact internal organs or through injection of the body cavities with fixative. Some diseases, such as anthrax (caused by the bacterium Bacillus anthracis) or Ebola viruses, cause serious zoonotic diseases that can be fatal in humans. The former is of particular importance during ruminant necropsies (though other wild and domestic ruminants, horses, zebra, wild pigs and carnivores can be infected2), the latter is of concern during great ape necropsies in parts of Africa. The most common gross finding in either disease can be bloody discharge from body orifices. If either or other life-threatening diseases are suspected upon encountering a carcass or during a necropsy, the necropsy should not proceed and the body should not be moved until testing to confirm or exclude its presence is completed. If possible, guarding the carcass to prevent scavenging is recommended. For anthrax, multiple cytologic preparations of blood (ear nick from the downside ear or coronary band), aspirate from the thoracic cavity, and/or tissue (submaxillary lymph node, spleen) should be air dried, fixed in methanol, and stained with polychrome methylene blue (McFadyean reaction) or Giemsa stain. The bacteria are large bacilli with square ends and a clear or pale halo; bacterial culture can also be performed for confirmation, but cytology will typically provide the quicker preliminary result. If positive, the necropsy should not proceed and the carcass should be burned and buried or buried.4,5

Outbreak Investigations As in all necropsies, a systematic approach is absolutely essential in managing the increased complexity, due to

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expanded scope and scale, that is inherent in an outbreak/ mortality event. In many countries, local and national plans or other strategies have been developed and are activated in outbreak situations. Familiarity with those that exist in your region is important because valuable resources that aid or are necessary in your response and investigation may be available through these programs. For example, the Incident Command System (ICS) is a management concept that was created to address the demands and complexities inherent in emergency response or natural disaster events. In the United States the ICS is used by many federal agencies, and its adapted use may provide organizational structure to veterinary outbreak and mass mortality event response and investigations. Main components of the ICS are: Unified Command, Operations, Planning, Logistics, and Finance and Administration; subdivision units (e.g., incident commander; safety, information and liaison officers) can be developed depending on the needs for the particular event (see https://training.fema.gov/EMIWeb/IS/ICSResource/ index.htm; accessed 16 Feb 2018). Critical components in mortality events and outbreak investigations include (1) identifying an investigative team and resources, (2) verifying that an outbreak is occurring, (3) establishing a case definition(s) and categorizing cases using results of necropsy examination, histology, ancillary diagnostic test results, etc., to establish the scope of the outbreak, (4) establishing baseline information about the event and disease (e.g., temporospectral, species, gender, age class), (5) examining the descriptive epidemiologic features of the cases to generate hypotheses, (6) testing hypotheses and performing additional analysis as needed, (7) implementing control measures (when possible), and (8) communicating findings and maintaining surveillance.12,13 For any event, it is also important to quickly determine if a disease (e.g., infectious, toxic) with significant zoonotic or animal health impacts (e.g., anthrax, Ebola) or illegal activities is present in order to engage and work together with the appropriate agencies and authorities in the mortality investigation. It is also important that the investigative team has a broad range of expertise (e.g., epidemiology, population biology, pathology, clinical medicine, genetics, bacteriology, virology, toxicology, and species- and environmental specific expertise) to help guide the investigation. Pathologists often work closely with epidemiologists during outbreak or mass mortality event investigations to construct case definitions, develop hypotheses on the cause of the event, analyze data, recommend ancillary diagnostic testing, and ultimately when making recommendations for control measures or long-term surveillance. Epidemiologic characteristics of an outbreak, along with historic data and necropsy findings, guide decisions related to first tier diagnostic testing (those deemed most relevant and immediately important). First tier diagnostic tests and histopathology findings often guide further ancillary testing. Approaches to necropsy evaluation and sampling during an outbreak or large-scale mortality event are like those used during routine, systematic, and thorough diagnostic

necropsies. Establishment of a sampling and necropsy protocol as soon as possible at the outset of the outbreak or mortality event is crucial to obtaining the best data to aid the investigation. Sampling and necropsy protocols should incorporate appropriate tissue collection for evaluation of not only the first tier differential etiologies but also a broad range of alternative etiologies. Often with large-scale mortality events, it is not practical or logistically possible to examine or sample every carcass in a reasonable postmortem interval. As such, focusing thorough diagnostic efforts on the freshest carcasses, especially those that die earliest in the event, may yield the most reliable data. In addition, plans should be established at the outset of the investigation for carcass management, database and sample management and storage, and sharing of data. It is also valuable during the course of the event to reevaluate the plan in order to make modifications based on what is being learned during the event (e.g., recognition of broader geographic scale or additional affected species) that increase efficiency and investigation outcomes.

Clean-Up and Carcass Disposal As occurs during a necropsy, PPE should be worn during clean-up and carcass disposal. Blood and residual tissues should be removed from instruments and work surfaces with soap and water, after which they should be disinfected. The best choices for disinfection have broad antimicrobial properties (e.g., 0.5% sodium hypochlorite; 10% bleach, 70% alcohol, borax; see also references [14 and 15] for more information about common disinfectants) and are effective against many common pathogens. Depending on the disinfectant (e.g., bleach), instruments may need to be rinsed after disinfection to prevent corrosion. It should be noted that prions are resistant to a number of common disinfectants and other forms of destruction.16 Current, best practice protocols for disinfection and carcass management should be obtained and implemented in consultation with governmental agencies for suspected or confirmed cases. Whether in a laboratory or a field setting, necropsy examinations and disposal of carcass, necropsy waste, sharps, and infectious materials should be performed in a manner that minimizes environmental contamination and exposure of the necropsy team or domestic or wild animals to infectious, toxic, or other disease agents. Efforts should be made to minimize the amount of nonanimal waste (e.g., aprons, masks, gloves, plastic bags) that is generated and burned, buried, or transported. It is important to be aware of and adhere to relevant organizational, local, national, and international regulations and protocols for carcass and medical waste disposal, which may involve consultation with local environmental and health agencies. Options may be limited due to a number of factors, including personal safety, location, size of a carcass(es), environmental conditions, location relative to water sources/catchments/wells etc., ability to safely move/relocate a carcass for disposal generally and within a reasonable timeframe (this may be logistically and politically complex and challenging),

CHAPTER 30  Wildlife Necropsy Primer

financial limitations, animal welfare and environmental considerations, and willingness/ability/permission of contracting facilities/landfills, etc. to accept the carcass/waste. Options for carcass, tissue, and waste disposal include incineration/burning and burial, burial, rendering, landfill, fermentation, and biocremation (alkaline hydrolysis), which may be possible on-site or by external contractors, as well as mounding, composting, and natural decay at the site of death/necropsy. A number of detailed descriptions of several of these options are available and include those of national and international governmental and nongovernmental regulatory agencies.4,5,17 On-site incineration/burning and burial or burial alone may be the most practical option in some cases (e.g., in mass mortality events). In known or suspected anthrax cases, incineration/burning and burial or burial, or other methods that prevent sporulation and destroy the bacteria, should be used.2 If prion disease is suspected, a 2001 report suggested rendering, incineration, and alkaline hydrolysis (all under certain conditions) as the most reliable technologies at that time for reducing the infectivity of the organism.4 In addition, remember that pathogens may persist, proliferate, and pose extended human and animal exposure risks in unburned, buried or closed plastic bags.

Sample Collection and Management Even when the cause of death seems obvious, it is important that interpretations and conclusions drawn during a mortality investigation on an individual or group of animals are informed by all relevant information. This process is sequential and iterative, with gross necropsy typically followed by histology. Both directly affect decisions about ancillary diagnostic tests that are needed to confidently arrive at conclusions and are subsequently performed. In addition to a thorough, systematic necropsy examination, collection of a set of tissues for histology and ancillary diagnostic testing and tissue archiving (Table 30.1) should be a standard in all necropsy examinations. It may not always be possible to collect all of the recommended samples from each animal, but the more consistently these goals can be achieved and reports generated, the greater the chance that we will accurately identify diseases and disease trends. Pathologists are often asked about the minimum set of tissues that should be collected or that are necessary to make a diagnosis. However, in situations in which collection of a complete set of tissues is not possible, a should be collected. This will vary by species and circumstance but should include those tissues that are most likely to be most diagnostically relevant (e.g., common sites of pathogenic infection). In general, these are the major organs and include heart, lung, liver, kidney, spleen, brain/spinal cord, stomach, and small and large intestine. Targeted tissue testing can also be performed for specific suspect diseases (e.g., brain, lung, tracheobronchial lymph nodes for suspected canine distemper testing; liver, kidney, fat, stomach content, spleen, hair, brain for general toxicology screening; urine for domoic acid testing).

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Collecting a limited set of tissues may provide a diagnosis. However, this may reflect an incomplete picture of the cause and contributing or predisposing factor(s) in death. In a worse-case scenario, this can lead to misinterpretations and false conclusions that drive inappropriate husbandry, medical management, or other animal- or outbreak-related activities and mitigation strategies. Sample handling and storage for histology and ancillary diagnostic testing are critically important to optimize their diagnostic value and contributions to animal health, management, surveillance, and conservation outcomes (see Table 30.1). With the exception of formalin-fixed tissues, most diagnostic samples need to be refrigerated or frozen to prevent degradation and maintain the viability of pathogens, toxins, etc. if immediate on-site testing is not available, for transport to an external diagnostic laboratory, or when archived. This often includes establishing and maintaining a cold chain, which can be particularly challenging in field settings.18 This involves appropriate, consistent temperatures using one or a combination of materials and equipment. Storage containers/equipment holding diagnostic samples should not be used for holding food, beverages, or other medical materials such as pharmaceuticals. Basic equipment typically includes refrigerators (4°C ice chests/boxes, ice/gel packs, etc.), freezers (e.g., 0°C, −20°C, −80°C; non “frost free”), and containers appropriate for standard and ultralow storage (e.g., cryovials). Other options include dry ice and liquid nitrogen (LN2) (e.g., dewars [vacuum flasks], dry shippers [vapor shippers]). Use of and handling samples stored at ultralow temperatures should be carried out with extreme caution because exposure can cause severe burns. In additional, dry ice and LN2 vapor can cause asphyxiation and can be explosive so should be used only in ventilated spaces or containers; handling should be limited to trained personnel. For electrical equipment, a consistent source of power is an absolute necessity. In all settings, backup sources of electricity, often in the form of a generator, are essential. In less-developed settings, multiple generators may serve as both the primary and backup power sources. Scheduled monitoring and documentation of temperatures should be standard operating procedures to ensure consistent and continual storage temperatures. Whenever possible, samples should be immediately stored and frozen samples handled in ways that minimize freeze-thaw cycles. Two significant sample management challenges are space and sample curation. With proper storage, tissues are a valuable diagnostic resource for many years. Managing space, cost, power needs, ventilation, duration of research projects, chain of custody, and other aspects of short- and long-term management of samples all need to be considered and strategies developed. Plans and protocols for the curation of sample storage and movement (e.g., for diagnostic testing, archiving, to fulfill research requests) must also be developed and implemented. Sample information has historically been maintained in paper logs; however, most people, even in remote locations, have access to computers and use electronic databases or software, which need to be

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TABLE 30.1  Diagnostic Tests and Recommended Sample Collection, Handling, and Storage

Diagnostic Tests

Sample Collection

Sample Handling

Sample Storage

HISTOLOGY‡: The microscopic examination of tissue to study the manifestation of disease. It is a valuable adjunct to gross observation because many diseases may look similar macroscopically.

• Collect samples from all organs and any tissue that looks abnormal (relative to adjacent tissue) • Collect a representative sample from each part of the organ/lesion • Limit sample thickness to 0.5 cm (1 cm3) for good fixation • For abnormal tissue, collect junction between normal and abnormal • Label important items or samples that came from a specific location • All samples from a necropsy may be placed in a single container

• Place tissue and 10% NBF in leak-proof container • Need 1 part tissue to 10 parts formalin for good fixation

• Short term: room temperature • Long term: room temperature DO NOT FREEZE

ELECTRON MICROSCOPY§: Higher magnification to resolve pathogens and tissue structure than may be achieved with routine light microscopy. Options include transmission, scanning, or negative stain electron microscopy.

• Tissue should be no greater than 1 mm3 • Common fixatives include glutaraldehyde or osmium tetroxide

• Place tissue into fixative (1 part tissue to 10 part fixative) in leak-proof container

• Short term: refrigerate • Long term: refrigerate DO NOT FREEZE

CYTOLOGY: The microscopic examination of fluids and cells. It may be performed immediately and aids in making a preliminary diagnosis. Cytologic review may occur locally or at a distant laboratory.

• Fluids: direct smear of fluid in thin film onto slide OR spin sample to make cell pellet (cell poor sample) that is then smeared onto a slide • Tissues: touch prep: gently blot on a paper towel to remove blood and then gently touch tissue to slide surface • Tissues: crush prep: collect small tissue sample onto slide; cover with coverslip/ cover glass; flatten tissue

• Fluids, touch prep: Air dry, fix (methanol), stain (e.g., Diff-Quik®, Gram, Wright Giemsa, acid-fast) • Crush prep: immediately directly view unstained tissue/cells

• Short term: clean, sturdy, crush-proof, bug/vermin-proof slide box or container • Long term: clean, sturdy, crush-proof, bug/vermin-proof slide drawer DO NOT EXPOSE AIR-DRIED SLIDES TO FORMALIN

MICROBIOLOGY: The study of microorganisms, including bacteria and fungi. Typical tests include bacterial or fungal culture or PCR. The most commonly sampled sites are obvious lesions (e.g., abscess) or the intestinal tract.

• Clean and sterilize sampling site (e.g., alcohol, heated blade) • Use sterile instruments/ techniques; minimizes risk of contamination • Instruments: swab, needle + syringe, scalpel blade, skin punch, trochar, etc.

• Place the sample/swab in appropriate medium for the desired test (e.g., bacterial culture media for aerobic or anaerobic bacteria bacterial culture)

• Short term (<1 week): room temperature OR refrigeration • Long term (>1 week): −80°C, liquid nitrogen (but may kill target pathogens) DO NOT FREEZE BLOOD OR SAMPLES FOR VIBRIO, LEPTO CULTURE

VIROLOGY: The study of viruses. Typical tests include viral culture or PCR. Commonly sampled items are lung, liver, spleen, lymph nodes, intestinal tract/ feces, brain.

• Clean and/or sterilize sampling site (e.g., alcohol, heated blade) • Use sterile instruments/ techniques; minimizes risk of contamination • Instruments: swab, needle + syringe, scalpel blade, skin punch, trochar

• Place swab/tissue in VTM • No transport media, no refrigeration available: place 1 part tissue: 5–10 parts 50% buffered glycerine

• Short term (<48 h): refrigerate • Long term: fresh tissue, VTM: −80°C or liquid nitrogen DO NOT EXPOSE SAMPLES TO DRY ICE REFRIGERANT

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TABLE Diagnostic Tests and Recommended Sample Collection, Handling, and Storage—cont’d 30.1

Diagnostic Tests

Sample Collection

Sample Handling

Sample Storage

PARASITOLOGY: The study of parasites and parasitism. Depending on the species, a wide variety of external (ectoparasites) and internal parasites (endoparasites) may be present. Collection allows for parasite identification and documentation. Some parasites also harbor infectious agents like bacteria and viruses and may be used for PCR analysis or virus isolation.

• Ectoparasites: Collect grossly visible parasites. Swab or scrape ears or skin for small parasites (may not be grossly visible) and smear material as a thin film onto a slide • Endoparasites: Collect grossly visible parasites. Collect fresh feces in a clean container or bag

• Place grossly visible parasites in 70% alcohol (ethanol) or formalin for identification • Examine scrapes under oil immersion microscopy immediately • Examine feces using standard procedures (e.g., gross appearance, direct smear, fecal floatation [for metazoan parasites, glycerin sedimentation for ameba])

• Short term: Fresh feces: refrigerate • Short and long term: Visible parasites: 70% ethyl alcohol or formalin; room temperature

TOXICOLOGY: Toxins are important agents to consider, particularly in the acute death of an animal or group of animals with few clinical signs. Toxins may be natural (e.g., poisonous plants, algae), agricultural (e.g., herbicides, pesticides, rodenticides), or industrial (e.g., lead, arsenic, waste from mining). Because many toxins are unstable and degrade with time, rapid testing or appropriate handling and short and longterm storage are essential.

• A large volume of fresh tissue is needed (50–500 g of sample) • Tissues useful in a general toxin screen: • Liver • Kidney • Brain • Stomach or intestinal content • Fat • Eye (aqueous humor) • Blood • Urine • Hair, flight feather

• Place tissues in clean, leak-proof containers with no chemical preservatives • Note: aluminum and plastic may leach and interfere with tests, so collection of two sets of tissues in different container types is recommended when possible • Filter paper (e.g., Whatman 903) for heavy metal analysis

• Refrigeration or freezing for transport is essential • Long term: −20°C, −80°C, liquid nitrogen

MOLECULAR DIAGNOSTICS: Typically performed for genetic or pathogen testing or discovery. Target tissues usually include: lesions and/ or liver, kidney, lung, spleen, brain, pancreas, gonads, lymph nodes, conjunctiva, mucocutaneous junctions of the oral cavity or genital tract, fluids/secretions, hair or skin.

• Small sample volume needed (1 g or less of tissue, 0.1 mL of blood or a clean dry swab) • Sample site should be clean and sterile • Wear gloves; use sterile instruments and techniques to collect samples and minimize risk of contamination • Instruments typically used for sampling: swab, needle and syringe, scalpel blade, skin punch, trochar

• Place sample in sterile container • Options: fresh frozen; VTM, RNAlater®, RNAlater-ICE, ethanol, TRIzol® (check preference of your lab, research project for others) • Blood or fluids: may be dried on a piece of filter paper (e.g., Whatman FTA for DNA-based infectious disease; Whatman 903 for proteins)

• Short term: fresh frozen: −80°C; RNAlater®: (<1 day) room temperature (37°C), (<1 week) refrigeration (4°C), (>1 week) −80°C; filter paper (<1 m) room temperature • Long term: fresh frozen, VTM, RNAlater®, filter paper: −80°C

*This list includes several common test categories and general recommendations for sample collection, handling, and storage. Specific best practice protocols should be consulted whenever possible, in development of your in-house standard operating procedures for sample management. † Recommended for labeling is pencil for glass slides, permanent marker or other appropriate inks for bags, tags, and labels that are resistant to alcohol, formalin, other solvents, refrigeration, ultralow temperatures (e.g., −20°C, −80°C, liquid nitrogen) routinely used in necropsy or cytologic procedures or tissue storage/ handling/transport. It is also useful, in addition to labeling the container/bag, to place a labeled tag (labeled ink) in the container; cut pieces of Tyvek® may be used. ‡ Tissues collected for histology are typically placed in 10% neutral buffered formalin (NBF). For NBF and tissues placed in other fixatives, it is critical to maintain a minimum ratio of 1 part tissue to 10 parts fixative to ensure proper fixation. Salinity and pH of fixative are important considerations for marine invertebrates. For invertebrates, 10% formalin or sea-water formalin are adequate fixatives in most cases. Specialized fixatives are preferred for some species such as zinc-formalin (Z-fix) for corals and Davidson solution for soft-bodied invertebrates (cnidaria). Decalcification can cause significant artifactual changes in the heavily mineralized tissues of some species, particularly corals and echinoderms. If the structure of the skeleton of these animals is critical for evaluation, the pathologist should consider submitting tissues for processing in a mineralized state (such as through plastic embedded and sectioning at a bone pathology laboratory) or a slow method of decalcification should be chosen (using the acetic acid in Davidson solution or pH-balanced EDTA). Enrobing the delicate tissues of coral species in alginate prior to decalcification has proven an excellent method for protecting the delicate surface polyps while retaining skeletal structural features. Formic acid and hydrochloric acid may be used for decalcification, but tissue disruption through gas bubble formation should be recognized and not interpreted as a lesion. For species with a chitin-based ectoskeleton, Pyreni solution may be used to soften the chitin and provide soft supple tissue for sectioning. § Recipes for marine invertebrate specific electron microscopy fixative are recommended. NBF, Neutral buffered formalin; VTM, viral transport medium.

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regularly updated and backed up to prevent catastrophic data loss. In some situations, for example, large active sample repositories or zoo collections with large sample inventories, dedicated staff may be needed to curate and manage sample acquisition and disposition information and databases, as well as sample retrieval and shipment. Archived inventories should be reviewed every 6 months to update additions and remove tissues that become redundant. Most facilities do not have the in-house resources or expertise to perform the broad range of tests that are run as components of a complete necropsy/postmortem examination. Shipment of samples to external laboratories is therefore a requisite of many necropsies. All sample shipments must comply with all local, state/provincial, federal/ national, and international laws and regulations to ensure human and animal safety and sample viability. Special packaging, handling, and labeling may be required (e.g., for shipments containing dry ice or LN2). For shipment to local laboratories, contacting the lab to discuss specific package labeling, submission forms, test requests, and minimum sample volumes is of value. For international shipments, export and import permits to ship samples and compliance with treaties and regulations concerned with the international movement of samples from endangered or managed species (e.g., Convention on International Trade in Endangered Species of Wild Fauna and Flora [CITES], US National Oceanic and Atmospheric Administration [NOAA for marine mammals], CDC [primates, rodents, civets, bats]) may also be required (see Chapter 4). The regulations and paperwork differ in every country and can be quite complicated, and processing can be excruciatingly time consuming. However, it is essential to understand all steps in the process, be in compliance, and pay attention to all of the many details. Failure to do so knowingly or unknowingly may result in significant fines, import/export permit refusal, confiscation and return of samples to their country of origin, or confiscation and destruction of samples by border patrol agents. Because of the latter, it is often a good idea, whenever possible, to maintain a duplicate set of samples at your facility as an assurance against problems that might occur during the shipping process. In addition, many diagnostic labs will discard tissues, glass slides, or paraffin blocks after a certain retention time, and many claim ownership of all submitted specimens (which they may or may not have the legal authority to claim). Written agreements with laboratories may be required to ensure return, retention, or ownership of your samples.

Data Management and Sharing All historical data, pathology reports, ancillary diagnostic test results, photographic documentation, and sample archiving and curation must be organized and managed. In many settings the records exist as/in a combination of paper and electronic documents and databases. However, technologic advances will likely push all activities related to documentation, even that done in the field, onto electronic

platforms in the not too distant future; robust systems for secure data storage and backups will therefore continue to be a necessity. These databases contain a treasure trove of baseline and disease-related information and available samples, all of which are hugely valuable in retrospective and prospective projects focused on wildlife ecology, health monitoring and disease surveillance, and conservation projects in zoos and other managed and free-range settings. Shared access to these valuable resources should be the rule rather than the exception (e.g., collaborative research projects, biomaterials requests, contributions to SSP/EEP and TAG programs). Whether retrospective or prospective, inclusion and participation of wildlife veterinary pathologists, who can appropriately interpret and contextualize data to prevent misinterpretation (a common problem in data review by nonpathologists) and contribute to and guide the development of protocols that involve necropsy and sample collection from free-ranging wildlife or zoo animals, are essential.

Communication Communication during a necropsy or active mortality investigation and after are critical, both practically and politically. Communications should be directly coordinated first with managers and/or managing authorities (institutional, local, national, etc.) before being more widely disseminated. Information is generally of interest and should be shared regularly and transparently with stakeholders (e.g., clinical veterinary staff, keepers, curatorial staff, collaborators, indigenous farmers, public) as results become available and interpretations and conclusions are drawn. Immediate communication of test results is also specifically important for medical treatment and/or mitigation of disease in contact with wild or domestic animals, staff, or others (e.g., public health networks) and for directing additional ancillary diagnostic testing. As mentioned previously, suspicion or confirmation of listed or reportable diseases must be immediately shared with appropriate local, national, and international authorities (see above), who may need to participate in disease management. The format for the communication varies depending on need and may include sharing of complete or summary pathology reports; peer-reviewed publications; presentations at professional conferences or community meetings; development or iterative revision of protocols, practical technical manuals, and other teaching materials; or other forms of communication with the media, including interviews and articles in the lay press. For highprofile cases or outbreaks with high public interest, it may be of value to identify a communications lead or team to field questions and provide comments that are informed by the pathologist and necropsy team. Creating a list of key talking points to be used by the communications team is beneficial to guide messaging, especially when conveying complex scientific information to the public. Care should also be taken to communicate only confirmed data or to qualify all results as preliminary until they are confirmed.

CHAPTER 30  Wildlife Necropsy Primer

Acknowledgments The author is extremely grateful to Drs. Alisa Newton (Wildlife Conservation Society) and Kathleen Colegrove (University of Illinois, College of Veterinary Medicine) for their contributions, especially on the topics of invertebrate necropsy and outbreak response, and critical review of this manuscript.

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