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CASE NOTES

Wildlife necropsy technique

Nicole Dobson, Karrie Rose, Heather Fenton and Jane Hall, Australian Registry of Wildlife Health, Taronga Conservation Society Australia

Posted Flock and Herd July 2023

Abstract

Over the past year the Australian Registry of Wildlife Health (the Registry) has taken on a more formal role leading wildlife disease investigations in NSW. Working closely with NSW Department of Primary Industries (DPI), Department of Planning and Environment (DPE), Environment Protection Authority (EPA), the Environmental Forensics laboratory, and a network of non-government groups, the Registry coordinates wildlife disease events that fall within agreed terms of reference. The Registry focuses on unusual or mass mortality events in free-ranging wildlife, monitoring for emerging syndromes, exotic pathogens to Australia, potentially zoonotic pathogens and threatened or culturally significant species.

We are grateful to the District Veterinarian community for your contributions to wildlife disease observation, reporting and sample collection as partners in the investigation pathway. We welcome members of the community to join us at the Registry to gain further experience in wildlife necropsy techniques. We are available and interested to deliver regional training programs and workshops for District Veterinarians, targeted veterinary practices, council, and EPA officers. Late last year, District Veterinarian Dr Phil Kemsley organised a wildlife disease investigation workshop for the Northern Rivers Wildlife Network where we were able to engage with local veterinarians and wildlife rehabilitators in the process and pathways of disease investigation. The workshop has resulted in greater awareness of the information and samples required for a fruitful investigation and higher quality sample and information submissions which have bolstered diagnostic outcomes.

Year in review

Since the last District Veterinarian's conference (5-7 April 2022), the Registry has examined 365 individual specimens representing 225 cases. The caseload is diverse, including 23 amphibians, 127 mammals, 32 reptiles, and 174 birds. These cases have largely emanated from coastal areas but represent wildlife across the state as illustrated in figure 1.

Map of NSW showing red dots
Figure 1 Wildlife specimen submission locations across NSW, 1 April 2022 - 31 March 2023

Forty-four of out 225 cases represented mass mortality events, including multiple flood-related mortalities in macropods, large-scale amphibian and grey-headed flying fox mortality events, malicious intoxications of birds, botulism and blue-green algae biotoxin events, psittacine chlamydiosis clusters, the investigation of an emerging skin disease in echidnas, and widespread outbreaks of necrotising enteritis in rainbow lorikeets associated with Escherichia albertii infection. Several wild animals were also examined on behalf of NSW DPE compliance as legal investigations pertaining to threatened species.

Notifiable disease exclusion is an important part of each wildlife mortality investigation, and a summary of our recent findings is included in table 1 below.

TABLE 1 NOTIFIABLE DISEASE TESTING CARRIED OUT 1 APRIL 2022 - 31 MARCH 2023 BY NSW DPI AND THE AUSTRALIAN MUSEUM

Number tested Number positive
Influenza A RT-PCR 54 10*
Newcastle Disease RT-PCR 54 0
West Nile Virus RT-PCR 29 0
Japanese Encephalitis Virus qPCR 12 0
Chlamydia PCR 9 3
Australian Bat Lyssavirus PCR 3 0
SARS-CoV-2 PCR 1 0
Batrachochytrium dendrobatidis 5 5
*Low-pathogenic only. Results from pooled samples, awaiting specification and typing

In addition to individual case management, the Registry has supported a number of DPI- and DPE-lead wildlife disease investigations, including:

To keep track of our wildlife diagnostic endeavours, please consider joining our Facebook group (Australian Registry of Wildlife Health) or visiting our website (arwh.org), review our bimonthly reports, or join one of our quarterly online updates. Quarterly updates are available to state government employees. Please contact safe address for ARWH for an invitation to future updates.

Please consider joining us next February (12-16, 2024) for a comprehensive course in wildlife health and disease investigation, featuring a day of skills-based workshops, followed by an intensive and extensive review of the diseases of each taxonomic group of wildlife. This Wildlife Health and Pathology Short Course will be followed by a one-day Symposium on Wildlife Disease Management (17 Feb 24). Registrations and student scholarship applications will be opening soon on our website.

Wildlife Necropsy Technique

Reporting wildlife mortality events remains unchanged in NSW. Any event of concern should be reported through the Emergency Animal Disease Watch Hotline 1-800-675-888.

When there is concern that the event may be the result of a suspected toxic compound, please ring the Environment Line on 131-555. Please take note of the EPA call number and include that on any specimen advice or sample submission form.

To enquire about necropsy technique, sample collection and submission, please ring the Australian Registry of Wildlife Health on 0481-468-505.

General Considerations for Wildlife Necropsy

Wildlife necropsies can be approached in a similar fashion to domestic species. The major points of difference emanate from the relatively limited knowledge, often multifactorial syndromes, and testing for the full range of disease syndromes across wildlife species. Thus, wildlife disease investigation often leans heavily on the histological examination of a full range of tissues, and the collection of a range of frozen samples to facilitate microbial culture, pathogen specific PCR, and toxicological testing. Since these investigations tend to be quite broad, they can be expensive, so starting with remains/carcasses that are as fresh as possible will maximise the value of the investment.

A typical wildlife necropsy includes a comprehensive history, photographs of the site and affected animals, a thorough external post-mortem examination, collection of ectoparasites and any lesions, and when appropriate, the collection of oral/conjunctival and cloacal swabs into separate PBGS containers. If a point source of a toxin is suspected, it is important to collect that from the site (i.e., any material that is likely an ingested bait). The skin is then incised top to tail to assess fat, muscle, and hydration prior to using clean instruments to open the abdomen and chest. Before touching any tissues, assess the tissues visually for exudates, parasites, or displacement. Then use another set of clean instruments to collect tissues into cryovials. Once your sterile tissues are collected, proceed with a thorough post-mortem examination, selecting representative tissues into 10% neutral buffered formalin. Don't forget eyes when they are fresh, and brain, spinal column, and peripheral nerve for neurological animals. Bone marrow in formalin is very useful for pale or anaemic animals. Collect larger samples of kidney, liver and gut content into individual aluminium foil wrapped packets and then a plastic container when intoxication is a possibility. Retain and freeze remains for a large event or when legal proceedings may follow. Additional documentation may be necessary for potentially legal cases, like cruelty investigations in domestic animals.

If live animals are available for examination, serum samples can also be very useful to have and archive. The Taronga Zoo maintains tissue, serum, and blood smear archives from many species from high-quality samples. Heart blood can be collected from animal remains for serum collection.

A typical sample set for a mass mortality event includes: photos, history, environmental samples (soil, water, food sources, potential bait material), morphometric data (weight, length and other data specific to the species), a full set of tissues fixed in 10% neutral buffered formalin, oral/conjunctival and cloacal swabs in PBGS for avian mass mortalities, sterile cryovials containing serum, ectoparasites, brain, liver, kidney and half of any lesions, larger aluminium foil wrapped packets of liver, kidney and gut content for toxicology.

When to initiate an investigation

Consider initiating a wildlife disease investigation when there is evidence of possible infectious disease such as:

When individual threatened or culturally significant species are found sick or dead, an investigation should be considered, as the findings may contribute to habitat or species management.

What to Bring

When you receive a report of sick or dead wildlife make sure that you go prepared to collect the animals. Environmental samples (such as water, soil, bait, or other foodstuffs), photographs and information. Animal remains disappear quickly in the environment.

Whenever possible, contact us at the Registry in advance of sample collection to ensure that appropriate materials and information are collected to answer your questions. But if you can't reach us, go ahead with gathering samples, photographs and information, as you may not get another chance.

Gear
Personal safety

Wear gloves when handling dead animals - vinyl, latex, or dish-washing gloves.

Wash your hands very well after handling animals (even if gloves were worn).

Wear a P2/N95 face mask for mass mortality events, marine mammals, parrots and if the animal displayed respiratory signs or aerosolisation of pathogens is likely.

Change your clothes before you contact live animals, food, or your children.

Be careful when handling, pouring, and transporting chemicals. Additional information regarding appropriate PPE, chemical safety and potential environmental concerns are available from the NSW Environment Protection Authority (EPA).

Only vaccinated and trained personnel with an appropriate titre level should handle bats (flying foxes and microbats).

Only trained personnel should handle dangerous animals such as monitor lizards and venomous snakes.

Be careful handling live animals. Beaks, teeth, and claws/talons can inflict considerable damage. If you don't feel comfortable handling a live animal, contact a local wildlife rehabilitation group to seek help.

Consider significant zoonoses including anthrax prior to handling or opening wildlife carcasses (see anthrax belt image available at www.dpi.nsw.gov.au )

In the event of a rodent plague, be cognisant of the potential occupational hazard posed by bird remains where zinc phosphide may be in use. Wild birds can store high concentrations of this material in their crops, which can be liberated as highly toxic phosphine gas: www.epa.nsw.gov.au

Field necropsies require additional health and safety consideration. Shade shelters, bug repellent (sprayed around remains and on gumboots), hats, drinking water, sunscreen are easily organised and make a significant difference to safety to comfort. Large animal necropsies are most safely conducted with a clean person taking notes, accepting samples, and keeping an eye on everyone's safety.

Record keeping

History, location, submitter details, species affected, clinical signs and timeline of events are incredibly valuable when investigating wildlife disease. Records should include morphometric data of affected animals whenever possible (weight, length, and species-specific data). Please refer to Appendix 1: Wildlife Incident Report Form, and Appendix 2: Morbidity and Mortality Log. These forms should be completed and submitted along with any samples or remains. Multiple species can be recorded on one form. The Morbidity and Mortality Log can be shared with landowners and wildlife rehabilitators to help track the event.

Photographs

Collect photos from a distance and close-up to allow others to see the environment surrounding the event, where samples, live animals and remains were collected, and how the animals were distributed. Place a scale and label in close-up images to identify the animal, date, and location. Ensure images are in focus and labelled to assist identification of point of interest. Keeping the background clean and holding the camera perpendicular to the specimen vastly improve the utility of the images. Additional resources on specimen photography are available on the Registry website: arwh.org

Collecting dead animals

Fish and amphibians decompose very quickly. Consider collecting sick live animals.

In any outbreak situation consider collecting a combination of sick-live animals and dead animals.

We can provide advice regarding methods of humane euthanasia, and methods for wildlife are summarised in the American Veterinary Medical Guidelines www.accc.gov.au

Wear nitrile, latex or vinyl gloves, coveralls and a P2 mask to handle animal remains. Also consider using a shovel, tongs, or inverted plastic bag to avoid direct contact with blood, bodily fluids or remains. Knot the bag tightly on itself or with a piece of string.

Bag each animal individually.

Use the masking tape to label each bag with the species (if known), date, location, and any animal identification (ear tag, microchip number).

Collect the freshest remains possible.

Decomposed remains with maggots can be useful for the diagnosis of botulism but contact the lab in advance of submitting these kinds of samples. Ensure that you collect these samples individually in tightly sealed bags to avoid contamination of other remains.

Collect as many fresh carcasses as possible. Aim for up to 20 small animals, and 5-10 medium to large animals.

Place protective equipment and any rubbish into your rubbish bag.

Wash your hands and boots thoroughly. Wash and disinfect the containers and all equipment upon return. Dispose of waste responsibly.

Environmental samples

Consider collecting water sources, food sources, soil, and any potential chemicals or materials that could contain chemicals. Wear gloves and safety goggles when appropriate. When poisoning or pollution is considered likely samples should be collected into glass jars provided by EPA.

Water samples are very important for the diagnosis of blue-green algae intoxication. Two water samples are required to adequately test for blue-green algae:

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