Necropsy of non-domestic animals and wildlife present interesting opportunities for the private practitioner both in areas of professional growth and disease investigation. It also provides challenges that can be as frustrating as they are rewarding. What follows are some tips to assist both the novice and seasoned exotic animal practitioner in post-mortem examination of exotic animals.
What the Heck is it?
It may seem like a simple task, but correct species identification is critically important and isn't always as easy as it seems. For example, many turtles and amphibian species can look alike and in some instances it can be difficult to tell common from endangered species. Would you recognize a bog turtle (Glyptemys muhlenbergii)? Would you be able to differentiate it from a spotted turtle (Clemmys guttata)? And would you know that bog turtles are the rarest of all North American turtles and that illegal possession (perhaps by your client) can result in fines or arrest.2 Indeed, local, state or federal laws and regulations that govern what and how the carcass or tissues from wildlife, endangered or migratory species or non-domestic animals in the pet trade are to be handled may affect the degree to which you can or should proceed with a necropsy. In New York, Peregrine falcons (Falco peregrinus) must be submitted to the New York Sate Department of Environmental Conservation for necropsy. In California, there is a dedicated pathology lab for sea otters. Also, it's important to be able to differentiate poisonous animals from their non-poisonous look-alikes, as is the case in coral snakes (Micrurus fulvius fulvius; poisonous) and scarlet king snakes (Lampropeltis triangulum elapsoides; non-poisonous). It may take a few minutes to key out the animal, but it will certainly be worth the time to make sure you know what you're dealing with. What you're dealing with may help a lot when it comes to what you should be thinking as it relates to the animal's death and your investigations.
Is that Normal
We've all learned about hearts and gizzards and hooves. Would you know where to find the Bursa of Fabricius and gonads in a young bird, the hemipenes, spleen and pancreas in a snake, the swim bladder and head kidney in different species of fish or the obex, tonsils and retropharyngeal lymph nodes in a ruminant suspected of a transmissible spongiform encephalopathy? How many thyroid glands are there and where are they located in turtles versus frogs? Is an iguana supposed to have fat bodies, and what if he/she doesn't seem to have them? Though most vertebrates have relatively common anatomical structures, there are variations in number, location and relative size of organs and some structures are quite cleverly species specific. Having several good anatomy texts and access to online databases provide great resources to help answer these and many other questions that you'll find yourself asking before (hopefully) or surely during (you know you're out there) the necropsy procedure.
Non-domestic animals are just bigger or funkier versions of their domestic counterparts. What's the problem?
In 2007 a young, experienced wildlife biologist died after contracting pneumonic plague from a free-ranging mountain lion (Felis con-color) he'd necropsied.3 Knowing the diseases that are common locally or have been reported in the species you necropsy will aid in protecting you and your staff as well as other animals in the environment (hospital or field sites) and may provide critical information for your health provider should you become ill after necropsying domestic or non-domestic animals. This knowledge will also act as an aid and guide during the necropsy since, in addition to the routine tissue collection procedure, you'll be on the look-out for certain diseases and be prepared to collect appropriate samples for routine and special ancillary diagnostics. Results of your necropsy procedure may reveal disease processes such as leptospirosis, rabies, psittacosis or rabbit hemorrhagic disease that are reportable to local, state and/or federal agencies. In these cases, do you have a reporting plan and do you have an established relationship with these agencies? If you do, your response will be controlled, even in what is often a stressful situation.
D and Judy's Top 10 Necropsy Tips
1. More is....well, more is more
We've all heard that the 'supersizing' of everything, in particular the food we eat, is killing America and that bigger isn't necessarily better. While that may be true under some circumstances, during a necropsy, more really does add value! Remember, it can be quite difficult, or even impossible depending on the method, to retrieve tissue samples from a carcass once it's been disposed of. During a necropsy, it's always a good idea to collect a complete set of tissues (see www.aazv.org for examples) and in some cases, two sets (e.g. one for the Species Survival Plans (SSP) pathologist). In addition, also consider cytologic preps (impression smears) of blood, body fluids, bone marrow or tissues; sterile collection and ultralow freezing or filter paper collection of feces or tissues samples (e.g. skin, skeletal muscle, liver, spleen, brain) for molecular diagnostics or genetics testing; swabs for bacterial, mycoplasmal or fungal culture; double sets of samples (e.g. fat, kidney, liver, brain, stomach contents, eye) in glass and foil for toxicologic testing; liths for content analysis and the always popular but often forgotten collection of feces and ecto and endoparasites for float or i.d.
2. What's it all about?
Though it may seem redundant, it's important to reiterate: The collection of a full set of tissues and samples is really important (see tip #1). We know how easy it is to collected limited tissues by being distracted by a big, juicy lesion. Even worse is when there is no good lesion to be found. In both instances, focusing on collection of a complete set of tissues (don't fall for that distracting fractured wing in the raptor without collecting tissues to rule out West Nile virus) will serve you well when it comes to putting the pieces together that explain an animal's clinical illness and death.
3. Where's Waldo?
Economics being what they are, thought it isn't optimal, it's recognized that is not always possible to collect a full set of tissues or submit all of the tissues collected at necropsy for histologic review. In situations driven by limited resources, be they your time or your client's pocket-book, go for the gold - focus on collecting the obvious lesion (with adjacent normal for comparison) or location likely to be responsible for the clinical signs. This may include collection of small organs such as the thyroid, adrenal or pituitary gland or the brain or spinal cord, all tissues that can be somewhat challenging to collect for first timers but that become easier with a bit of practice. If the sample is a whisper of tissue, consider placing it in a small vial or in a tissue cassette. Call your friendly pathologist for pointers if you're feeling at all intimidated by the tissue or its size.
4. How'd you get THAT in THERE?
In these times of "green" initiatives and a resurgence of recycling, re-purposing glass jars and containers for use as necropsy collection vessels seems one small but simple step towards a healthier planet. However, do keep in mind that fixed tissue samples don't come out of jars as easily as fresh tissues slither into them. A few tips to consider. Use jars that have wide mouths with screw top lids for ease of tissue removal and to prevent formalin evaporation during storage or leakage during transport. I'm sure there are many pathologist's out there that have had to figure out a safe and effective solution to removing a too big heart or inflexible limb from the too small opening of a glass ("guess I'll have to shatter it") or plastic ("gonna need the saw") jar. Also, as formalin has a penetration limit, trim tissues so they are not more than 0.5 cm thick and ensure 10:1, formalin to tissue volumetric ratio, which will ensure good samples for histology and will ease the transition from the storage container to the pathologist's trim table. The cost of formalin is inconsequential if skimping results in poorly fixed non-diagnostic tissue samples.
5. A picture's worth 1000 words
Good quality photos are a tremendous resource and when in digital format, they are immediately ready for sharing. They form not only part of the medical record but can be used in the creation of image archives (e.g. reference, research), as illustrations of descriptions included in biopsy or necropsy reports and can be easily sent to colleagues and specialists for consultation. Photographs are also important supporting documents in legal cases or forensics investigations and whole body shots from multiple angles are recommended in cases of this nature. Photo-documentation of every abnormal finding (make sure to label them appropriately!) is the gold standard so the availability of a second set of hands will make taking lots of photos during a necropsy infinitely more feasible! As a reminder, animal identifiers (owner/animal name), date and an objective measuring device (rulers rather than surgical instruments or currency) should be included in all images. Once a necropsy is over, you won't get a second chance to re-do the photos. Try to take high quality images with minimal blood, good white balance, and proper composition.
6. When the trickle becomes a flood!
The call may come from the breeder of exotic frogs, the mega pet store down the road or your local wildlife biologist. At first one was found, nothing too out of the ordinary. Now there's a pile of dead animal's with others sick and likely to follow. Unusual mortality events have no consideration for the time or your schedule. But the welfare of remaining and contact animals and potential public health concerns dictate the need for a speedy response and organized plan. Mobilization of staff or linkage with others responding to the same event will aid in early collection of tissues critical for diagnostic purposes; alerting your pathologist and diagnostic labs about the event will assist them in managing the increased volume that results from the event in a timely fashion. Communication within and among the various groups working on the event, with clear lines of reporting, and identified team leaders and spokespeople, will improve efficiency and decrease confusion. Local, state or federal officials may also need to be involved depending on the species involved or the known or suspected cause of the event.
7. The deepest sleep
There are probably few non-domestic and exotic animal practitioners who haven't been asked by a client or haven't had to determine the most acceptable method of euthanasia for a non-domestic animal. Whether a fish or frog or deer, recommended methods of euthanasia differ between different species (guidelines have been published by both the AAZV).1 When multiple options exist, it is suggested that a method with the least impact on tissues valuable for making gross or histologic diagnoses is selected. For example in cases of suspected or confirmed cardiac disease, intracardiac or intravascular injections of barbiturates can create significant artifacts in the heart, liver and lungs (especially if high volume: weight is used); acceptable alternative methods might include intramuscular or inhalant methods. Taking the necropsy into consideration prior to euthanasia will take some forethought and planning, especially if a client or the public will be present during the procedure. As a reminder, it is always valuable for the practitioner to communicate the method of euthanasia to the prosector and pathologist; in those animals immobilized (prior to euthanasia) with opioid analgesics such as carfentanil, it is critically important and a matter of personal safety.
8. When no means no.... and sometimes maybe
Interpreting histopathology results can be difficult. So what does it mean when your receive pathology reports that list "No gross or histologic lesions"? A diagnosis of no gross or histologic lesions doesn't mean that there are no clues as to what might have occurred as a lot can be learned from a negative necropsy result. If there is a good (well-collected, well-preserved), full set of tissues, the absence of lesions translates into there being no inflammation, degeneration, neoplasia or other identifiable pathologic process. It means that instances like management concerns, or toxic, metabolic or physiologic conditions or imbalances might be the basis for the animal's death. Sometimes, it means that the lesions aren't in the tissues examined. You may need to consider the possibility that there were lesions in samples that weren't collected or were collected but weren't submitted for review.
9. You can't always get what you want
On some days when the planets aren't properly aligned you might receive a pathology report that doesn't seem to make good clinical sense. We know, strange but sometimes true. When the results don't include tissues you know you sent or when the diagnosis doesn't match the clinical history or gross findings, there's likely to be a simple, rational reason more plausible than imagining the malicious efforts of a bored pathologist. This would be a good time to call and talk to your neighborhood pathologist to help in the realignment of the planets.
10. The party's over
Let's face it. You put off that necropsy until the end of the day so it wouldn't get in the way of casework. But you can't leave that mess, no matter how much you want dinner (your techs will kill you when they arrive in the morning!). Containers, bags, slides, swabs, and fluids need to be clearly labeled and stored so they can be retrieved and evaluated. Here's a quick tip that'll have you eating dinner and drinking piña coladas in no time at all. By placing all of the little bags and vials in a larger bag you can facilitate retrieval in the days to come (you'll need to label them then). Remember that fluids that need cytology need to be spun and the slides made before they are frozen. Reviewing photos can help when you're writing down your necropsy findings, but a quick set of notes will be invaluable in the morning when you transfer your notes into the medical record. If there are tissue issues, you may need to plan to hold the carcass remains for the next 2-4 weeks. Think ahead before closing the freezer door.
Ancillary Diagnostic Resources:
Microbiology: Routine aerobic and anaerobic cultures can be performed by most labs. But bacteria like Mycoplasma, Mycobacteria, Chlamydia, Brucella, and rickettsia all require special media and culture conditions. Bacteria like Salmonellas require diagnostics beyond culture to determine species. Molecular techniques can sometimes be used to identify unusual bacterial species or to identify bacteria that are not cultured in routine methods. Contamination with multiple bacterial species can impede these investigations. Many fungi are difficult to culture. Molecular techniques work well for these diagnoses.
For microbiology diagnostics consider: NVSL, your state diagnostic lab, the Fungus Testing Laboratory (http://strl.uthscsa.edu/fungus/), Myco-bacteriology Laboratory at National Jewish Health (http://www.nationaljewish.org/research/clinical-labs/about/learn/mycobac/index.aspx), E.coli reference Center (http://ecoli.cas.psu.edu/).
Virology: Viruses can be identified by visualization (EM), virus isolation (culture), and molecular technologies. EM requires well-fixed samples preferably fixed with glutaraldehyde. Virus isolation is often best performed on fresh chilled tissue. Because virus isolation relies on good samples placed on compatible cell lines, this can be very complex for exotic species. Molecular technologies are generally non-species specific but sometimes unaffected controls are needed to sort out results for affected animals
For virology diagnostics consider: NVSL, your state diagnostic lab, or labs with specific molecular diagnostic tests like UG Infectious Disease lab, Esoterix (http://www.esoterix.com/), and lots of university research labs!
Toxicology: Blessedly non-species specific. But there is no universal "toxin screen". Call your state diagnostic lab and discuss options and considerations. Sometimes pathology suggests which toxicology is indicated - sometimes not. Sometimes, the toxicology needs to be performed on feed or environmental samples. If intoxication might be a possibility, try to collect many samples close to the time of onset of illness or death.
For toxicology diagnostics consider your state diagnostic lab.
References
1. American Association of Zoo Veterinarians (AAZV). 2006. Guidelines for euthanasia of non-domestic animals. Pp. 111
2. Hulse, A.C., C.J. McCoy and E.J. Censky. 2001. Amphibians and Reptiles of Pennsylvania and the Northeast. Cornell University Press, New York. Pp. 419
3. Wild, M.A., D. Wong, C. Higgins, J.G. Powers, T.R. Spraker, E. Perez, P. Mead, and A. Chanlongbutra. 2008. Improving guidance for safe handling of wildlife: Insights from a wildlife biologist's death. Proc Wildl Dis Assoc, Edmonton, Alberta Canada. Pp. 23