Wildlife Forensic Pathology

Abstract

Global environmental threats present a challenge to scientists and the public alike. Both the disappearance of species and the accompanying decline in biodiversity urgently require a multidisciplinary and interdisciplinary scientific approach. This in turn warrants the application of techniques historically restricted to human and veterinary medical diagnosis or forensic investigations. In particular, there exists an unprecedented opportunity for input by pathologists and the appropriate use of their modus operandi, including necropsy and the laboratory examination of samples. The application of pathological methods to wildlife work is outlined, with particular reference to the investigation of crime and the emergence of conservation forensics as a subject in its own right.

“Kipya kinyemi ingawa kidonda”

(Swahili Proverb—a new thing gives pleasure, even if it is a wound).

Appendices

Appendices

Appendix 1: Forensic Examination of Live Animals: Birds

• 1. Remember that observation of the bird and its environment should precede restraint, handling or clinical examination. Note the bird’s behaviour, including commenting on how docile, tame or habituated it appears to be, whether it might be imprinted, whether it is easily frightened by noises or other stimuli and whether it responds normally to routine stimuli. Familiarise yourself with the natural history and biology of the species which is the subject of the investigation. Consider involving a colleague, bird-keeper or ornithologist, who may have more knowledge of such aspects than you do.

• 2. Carry out as full an investigation as possible. In addition to standard clinical examination record weight (mass) and standard measurements, give a condition score and report specifically on the following: plumage (including evidence of moult or pinioning), beak, claws, soles of feet, presence/absence of leg rings (bands), jesses, telemetry equipment or other attachments, presence of tattoo(s) or coloured dyes. Pay special attention to clinical signs or lesions that may be relevant to a history of alleged neglect, cruelty or persecution Throughout the clinical examination, practise strict hygiene, following a proper Risk Assessment.

• 3. Take photographs throughout the examination, even if only simple photographic equipment is to hand, especially if there are lesions that may change in appearance if there is any delay.

• 4. As a routine, take the following samples for laboratory tests: fresh droppings (faeces and urates), blood smears, blood for haematology/clinical chemistry, dropped feathers, pellets/castings (where available) and ectoparasites. Mark all specimens carefully and fully, with the label on the container, not on the lid.

• 5. Depending upon the circumstances, be prepared to carry out supporting investigations, e.g. radiography and ultrasonography, emesis and lavage. Imaging may reveal significant lesions that cannot be easily detected on palpation.

Some Further Reading

• Cooper JE (2002) Birds of prey: health & disease. Blackwell, Oxford, UK

• Cooper JE, Cooper ME (1986) Is this eagle legal? A veterinary approach to litigation involving birds. In: Proceedings of the Forensic Zoology Discussion Group. Zoo-technology, London, pp 27–30

• Cooper JE, Cooper ME (1991) Legal cases involving birds: the role of the veterinary surgeon. Vet Rec 129:505–507

• Cooper JE, Cooper ME (2013) Wildlife forensic investigation: principles and practice. Taylor & Francis/CRC Press, Boca Raton, Florida

Appendix 2: Submission and Report Forms

Appendix 3: Examination of Eggs and Embryos

Appendix 4: Turtle Egg/Embryo Examination Form

Appendix 5: Field Equipment Lists

Equipment—General Items

Recommended equipment is listed below:

• Protective clothing including boiler suits (coveralls), gloves (surgical, thick kitchen, long rubber etc.), masks, goggles, boots and overshoes.

• Barrier tape, flags, markers, cones and other crime scene security items.

• Protective gloves and clothing, barrier tape and tools, such as tongs and hooks for handling hazardous materials.

• Disinfectant and deodoriser to neutralise smells from carcases etc. (both used with caution at crime scenes as they may destroy trace evidence).

• Collection kits (prepacked) for taking samples for laboratory investigation, including toxicology, DNA analysis and haematology.

• Appropriate labels, tamper-proof tags, evidence seals etc.

• Marking pens.

• Scales/balances, callipers and micrometer for weighing animals, tissues and samples.

• Rules, tapes, centimetre scales etc. for measuring.

• Equipment for taking casts of dentition, imprints in bones, animal tracks etc., including plaster-of-Paris, other powders and silicone-based materials, waxes, plus retention frames or stiff cardboard.

• Trace-evidence collection equipment.

• Evidence packing, e.g. bags, boxes, tubes, envelopes and other supplies for packaging and storing evidence.

• Photographic kit, including (as appropriate) digital/still cameras, video cameras, magnifier, night vision equipment, aerial camera system and camcorder.

• Binoculars/field glasses.

• Torches (flashlights).

• Blue-Light kits and supplies.

• Egg candler.

• X-ray viewing screen.

• Magnifying glasses/hand lenses.

• Magnifying loupe or dissecting microscope.

• Clipboards and record sheets, plus pens and pencils.

• Chalk and crayons.

• Elastic bands and string.

• Tape-recorder (preferably voice-activated) and tapes.

• Evidence seals/tape.

• Kits and systems for collecting and preserving delicate crime scene evidence (see text).

• Computer and appropriate software, e.g. barcode tracking systems for property, evidence and crime scene reconstruction.

• Handheld computer.

Equipment: When Working in the Field

The detailed contents of field kits will depend upon the location and the type of investigations being carried out.

The following items are recommended in the field for (a) clinical, (b) post-mortem and (c) laboratory diagnostic work, respectively. In each case, the list should be supplemented with general items, as above.

• (a) Clinical equipment—when live animals have to be examined

  • Stethoscope (lightweight)

  • Auriscope (otoscope) (lightweight)

  • Ophthalmoscope (lightweight)

  • Rigid endoscope (battery-operated)

  • Pen torch (flashlight)

  • Spare bulbs and batteries

  • Syringes and needles (disposable)

  • At least one boilable, reusable syringe and needle

  • Empty drinks cans, labelled “sharps boxes”, for used needles, scalpel blades etc.

  • Disinfectant(s), including ethanol

  • Camping (gas cylinder-operated) stove—for sterilising, lighting and cooking

  • Pressure cooker for sterilising

  • Selected medicines, including local analgesics, sedatives and agents for euthanasia (plus gun if large animals may need to be killed)

  • Cotton wool

  • Dressings

  • Suture materials

  • Basic surgical (“cut-down”) set and other instruments as necessary

  • Disposable skin biopsy punch

  • Cautery (battery operated)

  • Clippers for claws, talons, beaks

  • Ring (band) remover

  • Spring balance(s) or battery-operated scales

  • Cloth bags and other devices for restraining small animals

  • Gloves—surgical and for handling

  • Towel

  • Oesophageal and other tubes

  • Mouth gag/wooden spatulae

  • Aluminium foil

  • Sampling and other equipment for laboratory work (see later—list (c))

• (b) Post-mortem equipment—when dead animals have to be examined

  Standard necropsy items—portable/folding, lightweight/plastic where appropriate.

  • Saw(s)

  • Scalpels and blades

  • Knives

  • Forceps

  • Probes—solid and flexible (rubber)

  • Scoops for brain etc.

  • Pen torch (flashlight)

  • Spare bulbs and batteries

  • Syringes and needles (disposable)

  • Empty drinks cans, labelled “sharps boxes”, for used needles, scalpel blades etc.

  • Disinfectant(s), including ethanol/methanol/methylated spirits

  • Camping (gas cylinder-operated) stove—for sterilising, lighting and cooking

  • Pressure cooker for sterilising

  • Cotton wool

  • Spring balance(s) or battery-operated scales

  • Scalpel handle and disposable sterile blades of several sizes and shapes. Dissecting scissors, curved haemostatic forceps, toothed and smooth-jawed fine-pattern thumb forceps and bone forceps

  • Sampling and other equipment for laboratory work (see later—list (c))

• (c) Laboratory equipment

  • Microscope (solar or battery operated)

  • Immersion oil (or methyl salicylate) with swabs and xylene for cleaning

  • Pre-cleaned, frosted, ground-ended microscope slides and slide box or tray

  • Pencils for marking glass slides

  • Diamond-tipped pen for marking glass slides (if frosted not available)

  • Worm-egg counting slide

  • Coverslips

  • Lens tissues

  • Saline, saturated NaCl solution and other reagents and equipment for parasitology

  • Transparent polythene strips and methylene blue/malachite green for the KATO method of cleaning faecal films for parasites, ova and cysts

  • Fixatives—alcohol, formalin

  • Selected stains for cytology

  • Lightweight (plastic) staining jar or staining rack

  • Urine and blood chemistry test strips

  • Portable centrifuge

  • Polypropylene capillary tubes, some coated with heparin or EDTA, plus commercial haemoglobin and PCV reader

  • Handheld refractometer

  • Transport medium for bacteria, viruses, mycoplasmas and Trichomonas

  • Vacuum flask

  • Buffer tablets for use—with local water

  • Scalpel, scissors, forceps, artery forceps (haemostats)

  • Wash bottles for alcohol, stains etc.

  • Lightweight pots for specimens

  • Disinfectant(s), including ethanol/methanol/methylated spirits

  • Camping (gas cylinder-operated) stove—for sterilising, lighting and cooking

  • Pressure cooker for sterilising

Recommended additional items when working overseas in the field

Sharp and other possibly dangerous items should not be placed in hand luggage when travelling by air, or through land or sea security checkpoints.

• Emergency pack containing business cards, letters of authorisation, protocols for snakebite (etc.), medicines and antidotes.

• Multipurpose Swiss Army-type pocket knife.

• Sewing kit with assorted needles, thread.

• Screwdrivers, pliers and an adjustable spanner.

• Elastic bands, string, dental floss, suture material, adhesive tape, insulating tape, duct tape, electrician’s tape.

• Spare nylon cable ties for securing lid hasp of case during travel.

• Standard veterinary and other textbooks: where space is limited, the “Merck Veterinary Manual” is recommended (but caution must be exercised in quoting from this in court as it does not include references).

• Phrase books of appropriate languages.

• The “SAS Survival Guide” (Wiseman 1993), which contains much useful information that can be applied to difficult situations in the field.

• Appropriate clothing, e.g. the shoulders should be covered when working in a Moslem community, a tie is a courtesy in most countries when a man is meeting dignitaries.

Additional Equipment for Specific Investigations

Blood sampling from live animals for DNA and other investigative purposes

• Syringes (1 ml, 2 ml, 5 ml/10 ml) as appropriate

• Needles (20 g, 25 g, 22 g, 28 g) as appropriate

• Anticoagulant serum tubes

• Cotton wool

• Frosted glass slides and box or tray

• Coverslips

• Pencils for marking glass slides, plus sharpener

• Methanol

• Cards or solutions for blood collection

• Magnifying glass/hand lens

• Plastic bags and labels

• Electronic/spring balance

• Ruler, tape measure

• Clipboard, black pen and record sheets

• Basic clinical equipment

• Restraining equipment—nets, towels, gloves, hood, bags

Plus other items from earlier lists, as necessary

Entomological (invertebrate) collection

• Small ventilated plastic vials, lined with mesh, filled with filter paper (to reduce condensation and subsequent drowning) for holding live ticks or other invertebrates.

Relevant identification keys and texts

More specialised laboratory investigations in the field

• Vacuum flasks and portable, lightweight, cool box.

• Normal (isotonic) saline.

• Hypertonic NaCl or sugar (sucrose) solution for flotation/sedimentation examination.

• Tincture of merthiolate for staining faecal protozoa.

• Fixatives for blood and other body fluids, bone marrow and endo- and ectoparasites.

• Rapid acting stains for blood and other body fluids (sputum, urine, synovial and coelomic, cerebrospinal, bone marrow etc.) and touch/impression smear cytology.

• Gram, acid-fast and other special stains.

• Lactol-phenol cotton-blue for demonstrating fungi.

• Plastic pipettes.

• Slotted stain jar. Lightweight, unbreakable, plastic staining jars are preferable to heavy, fragile, glass Coplin jars.

• Mounting media for permanent preparations of blood and bone marrow films.

• Clearing and mounting media for small ectoparasites.

• Light-weight, slide-drying rack.

• Transport media for bacteria, viruses and protozoa (see earlier).

• Microbiological test strips.

• Urine and blood chemistry test strips.

• Rapid diagnostic test strips.

• Cardboard strips, which can be labelled in pencil or waterproof ink, and placed inside specimen containers.

• Safety matches, a small Bunsen burner or disposable butane cigarette lighter.

• Squeeze bottles for methanol etc.

• Specimen containers, filled with concentrated formaldehyde, for dilution with river or seawater.

• Tongue depressors, wooden applicator sticks and sterile cotton-tipped applicators. Plastic coffee spoons for use as spatulae.

• Non-lubricated condoms as finger covers.

• Plastic film canisters (pots) with labels attached for faecal collection, parasites etc. They usually can be obtained gratis from film dealers or photofinishing laboratories.

• Sterile disposable venous and urethral catheters; latex or plastic tubing.

• Plastic slide boxes, each prefilled with polished, frosted and glass microscope slides.

Checklist of battery-operated or solar-powered (direct sun or solar panels) equipment that can be used in isolated locations

• Miniaturised otoscope (auriscope)

• Ophthalmoscope

• Rigid endoscope

• Colorimeter

• Electrocautery equipment

• Blood pressure monitoring instrument

• Minicentrifuge

• Miniphotometer

• Respiratory monitor and pulse oximeter

• Refractometer

Further Reading

• Cooper JE (2013a) What is fieldwork? J Exotic Pet Med 22(1):7–16

• Cooper JE (2013b) Field techniques in exotic animal medicine. J Exotic Pet Med 22(1)

• Cooper JE, Cooper ME (2007) Introduction to veterinary and comparative forensic medicine. Blackwell, Oxford

• Cooper JE, Cooper ME (2013) Wildlife forensic investigation: principles and practice. Taylor & Francis/CRC Press, Boca Raton, Florida

• Cooper JE, Cooper ME (2017) Appendix 3 Field pathology. In: Cooper JE, Hull G (eds) Gorilla pathology and health: with a catalogue of preserved materials. Elsevier, St Louis

• Cooper JE, Cooper ME (2017) Appendix 4 Hazards, including Zoonoses. In: Cooper JE, Hull G (eds) Gorilla pathology and health: with a catalogue of preserved materials. Elsevier, St Louis

Appendix 6: Samples

Mistakes can occur at various stages of sampling:

• Selection

• ↓

• Taking

• ↓

• Packing

• ↓

• Transportation

• ↓

• Reception

• ↓

• Processing

An error or inconsistency introduced at any stage in the sequence above can adversely affect subsequent stages and easily prejudice results.

Selection of Samples

In forensic work, this depends upon the circumstances and the questions that are being asked. The choice of sample will be influenced by the purpose for which the test is to be performed. Some investigations and procedures may be routine, others more complex. Samples for toxicology often present particular challenges and it may be wise to consult the laboratory before taking such material.

Samples are not only taken for biomedical investigation. Some, such as explosives, ammunition and weapons will be examined by specialists in forensic and other laboratories.

Some samples can be taken from either live or dead animals but others may only be realistically obtained from one or the other.

Table 7 Samples from live and dead animals

Table 8 Samples from animal remains

Plastic forceps are important. They are used for a variety of purposes, including the handling of lead shot, pellets or bullets where metal instruments may damage the surface, making identification of the weapon less easy and the taking of samples for analysis of certain metals.

Samples for histological examination are usually fixed in buffered formol saline (BFS). Lung, liver, kidney and any abnormalities should be taken routinely.

Samples for toxicological assay (blood, urine, tissues) should be placed in separate containers; this is different from histological or electron microscopic examination where tissues may, if necessary, be placed in the same container of fixative.

Correct, secure, storage of material taken from a forensic case is vital—as is confidentiality. Samples must be handled carefully because of health and safety considerations and labelled or marked so that they cannot be misplaced or transposed.

Legal aspects

The collection, transportation and processing of samples are likely to be covered by legislation, including Post Office Regulations, health and safety, animal health (see Cooper ME, Wildlife Conservation Law, this volume).

Appendix 7: Post-mortem Examination Form

Appendix 8: Specimen Forms—Wildlife Forensic Cases

Appendix 9: Summary of Post-mortem Findings