See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/308308465

Challenges and Solutions in Diagnostic Pathology of Exotic Small Mammals

Conference Paper · October 2015

CITATIONS READS

0 156

2 authors:

Rita Mcmanamon Drury Robb Reavill

University of Georgia Zoo/Exotic Pathology Service
22 PUBLICATIONS   189 CITATIONS    148 PUBLICATIONS   903 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Characterization of hepatic copper accumulation in ferrets with and without hepatobiliary disease and measurement of copper levels in 7 popular ferret diets View
project

Mast Cell Tumors in Iguanas View project

All content following this page was uploaded by Drury Robb Reavill on 19 September 2016.

The user has requested enhancement of the downloaded file.

 Close this window to return to IVIS
http://www.ivis.org

Proceeding of the
ACVP Annual Meeting

Oct.17-21, 2015
Minneapolis, MN, USA

Next Meeting:

Dec. 3-7, 2016 - New Orleans, LA, USA

Reprinted in the IVIS website with the permission of the ACVP

Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

Challenges and Solutions in Diagnostic Pathology of Exotic Small

Mammals
Rita McManamon, DVM1 and Drury R. Reavill, DVM, DABVP (Avian, Reptile & Amphibian
Practice), Diplomate ACVP 2
1
Zoo and Exotic Animal Pathology, Infectious Diseases Laboratory and Department of
Pathology, University of Georgia, Athens, GA 30602 (ritamcm@uga.edu); 2Zoo/Exotic
Pathology Service, Carmichael, CA (DReavill@zooexotic.com)

Introduction

Applying standard diagnostic anatomical and clinical pathology techniques and criteria to
exotic small mammal species is professionally satisfying, but also can be challenging. Our
diagnostic pathology practices serve a wide range of small and large zoos, aviaries with
mixed species enclosures, pet stores, and private practice veterinary clinicians; our patients
encompass a wide range of exotic large and small exotic species. Through these
experiences, we have developed or adapted necropsy and other diagnostic techniques for
these species, or have encountered some diagnostic challenges and tips from which other
diagnostic pathologists can benefit. This presentation selects a few common or challenging
diagnostic issues encountered with exotic small mammals, focusing on small “pocket pet
species” (such as rodents, rabbits, sugar gliders, hedgehogs), and ferrets. The selected
topics include: general necropsy planning to ensure ideal sample collection and submission;
sample submission for ancillary testing including immunohistochemistry; recent findings in
distinguishing inflammatory bowel disease from intestinal lymphoma in ferrets; and
commonly encountered conditions in sugar gliders.

Necropsy sample collection, submission, and temporary archiving

Relatively infrequently, the whole body of a deceased small exotic mammal is presented to
the diagnostic pathologist. Despite the negative consequences of potential autolysis, this
situation presents the best opportunity for the pathologist to perform optimal necropsy photo
documentation, sample collection, and freezing of fresh tissue samples for potential future
ancillary tests. But more often, the diagnostic pathologist receives a bottle of selected fixed
tissues from the clinician’s necropsy. This latter situation has the best chance for preserving
diagnostic tissue quality quickly, but relies heavily on the clinician’s selection of tissues.
Also, optimal selected fresh tissues or swabs may (or may not) have been frozen. Whenever
possible, pre-consultation between clinician and pathologist regarding the approach to a
routine necropsy, what routine tissues should be taken for which species, and consideration
of ideal sampling for suspected diseases of concern (particularly if there is any potential for
zoonotic disease transmission) often greatly improve documentation and proper sample
collection.

Diagnosis of some selected diseases often requires ancillary testing, beyond routine H&E

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

histopathology slides. For instance, experience shows that the best sample/test to be
ordered, to document the presence or absence of Streptobacillus sp. in the oral cavity of a
rat (due to suspected rat bite fever in a human) should involve PCR testing of a fresh swab
of the oral cavity – and not culture of the tissues (as the clinician might suspect).
Streptobacillus moniliformis is an extremely fastidious organism that has complex culture
requirements1. Appropriate fresh/frozen tissue samples need to be held in cases of
suspected Salmonella sp., methicillin-resistant Staphylococcus aureus, or other bacterial
infections in order to confirm links to human disease2. This also requires the submitting
veterinary clinician to provide a complete history and express concerns for zoonotic disease,
to the pathologist. Clients sometimes erroneously assume that routine histopathology will
reveal or refute all possible diseases even if they are not mentioned in the history. Correct
identification of the animal is also important. Prairie dogs (Cynomys sp.), and Gambian giant
rats (Cricetomys sp.) carried monkeypox to humans in contact with the rodents. Fresh
frozen lung samples appear to be best for molecular studies which are essential in
determining the specific genetic signatures of monkeypox virus3. Likewise, freezing samples
of kidney may be a more desirable sample than brain, if Lymphocytic Choriomeningitis Virus
(LCMV) infection is suspected in a rodent. Keep in mind that this is a highly hazardous
disease agent; it is important to contact the laboratory before sample submission for specific
sample preparation/shipping requirements. Another example of multiple tests required to
document a pathogen is one study which demonstrated that histopathology cannot be
considered sufficient to detect Encephalitizoon cuniculi, and ancillary tests such as PCR and
immunohistochemistry (IHC) are often needed4.

If a regular client relationship is established, using a written checklist for routine

formalin-fixed, fresh tissues and swabs can optimize sample collection. In our experience,
potentially useful tissues which are often overlooked by clinicians during gross necropsy
collection include: eyes, fixed samples of nasal turbinates in rodents (especially rats and
mice), bone marrow, muscle, and skin. For example, Encephalitozoon cuniculi can be
responsible for cataract and lens-induced uveitis in rabbits5. Nasal turbinates may harbor
sinusitis or abscesses. Bone marrow is useful to show whether myelosuppression or
myeloproliferative disease is present, in any species. Benzimidazole toxicosis in rabbits
results in bone marrow suppression6. If anemia is noted clinically, the hemogram (including
hematocrit and morphology of the erythrocytes) must be provided, along with collection of
fresh bone marrow smears for cytology, and fixed samples of bone marrow, for adequate
interpretation7. Samples of muscle and esophagus are important for the diagnosis of
disseminated idiopathic myofasciitis of ferrets8.

The challenge of how long to save formalin-fixed wet tissues, and frozen tissues, is a
constant struggle for space and prioritization. The best solutions are achieved through
proactive consultation about expectations, and clear timelines for disposal. At the very least,
temporary holding of tissues until the final histopathology report is issued and read, is
desirable. Holding of additional tissues and swabs are particularly helpful in cases where
concerns from the client regarding potential zoonotic disease transmission are not raised
until after the report is received. (More details regarding the diagnosis of zoonotic disease,

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

and teamwork between diagnostic pathologists in human and veterinary fields, are covered
in another talk during this session).

Challenges with immunohistochemical (IHC) tests in some species

Ancillary immunohistochemical tests, performed on tissues prepared from

paraffin-embedded blocks, can be extremely valuable in the diagnosis of infectious diseases
or in the identification of cell biomarkers for detailed tumor classification. In most cases, the
major limitations to the use of immunohistochemical stains lie in the choice and availability
of antibodies and the preservation of epitopes9, and special techniques may be necessary to
improve detection of agents or biomarkers4,9. Laboratory-specific issues such as quality
control, technique and experience of the operator, and age of the reagent used may also
influence success10. One critical factor, which may not be apparent to the primary diagnostic
pathologist when ordering an immunohistochemical stain, is to remember that the primary or
secondary antibodies used for these tests are typically produced through immunization of
mouse, rabbit, guinea pig, or human. In most cases this may not matter, but if the tissue
being examined is from the same species, the reagent will bind to endogenous sites within
the tissue10,11,12. When mouse, rabbit or guinea pig tissues are being examined, it is
important that the diagnostic pathologist consult with the laboratory, to find out what
reagents are being used, and to explore options. In our experience, many mouse-derived
antibody reagents are still useful. In a few cases, the laboratory may be able to vary the
techniques slightly and improve results. But in many cases, the only solution is to identify
and employ a different reagent, derived from a different species, and this may increase the
time involved and final cost to the client. In all cases using immunohistochemical stains on
non-routine species, one cannot assume that the reagent will work adequately. It is
important to validate the test result through positive and negative controls using normal
tissues from the same species, or through comparison with internal control tissues on the
same slide.

Distinguishing inflammatory bowel disease from lymphoma in ferrets

Inflammatory bowel disease (IBD) is a common intestinal syndrome which is identified in

ferrets13. The diagnosis of IBD is a clinical diagnosis, not a morphologic diagnosis (and this
distinction can be confusing to clinicians). Establishment of the diagnosis usually relies on
histopathological confirmation of severe inflammation, either from full-thickness intestinal
biopsies taken during exploratory laparotomies, or through endoscopic intestinal biopsies.
However, diagnosis can be complicated and frustrating, for clinicians as well as
pathologists. Clinicians are sometimes frustrated by a lack of consistency in morphological
diagnoses among pathologists, and pathologists are impacted by several factors:

• There are no currently established criteria for how many samples to take, and from
which sections of small or large intestine.
• There are no currently established criteria for this species, to histologically grade
severity of inflammation.

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

• IBD is a diagnosis of exclusion, by ruling out lymphoma.

• The spectrum from severe inflammation to intestinal lymphoma (which is also
common in ferrets) is sometimes unclear.
• Interpretation of inflammation may be made by pathologists with varying experience
with evaluating ferret tissues
• Variation in cell populations from samples submitted from different anatomic locations
in the same animal, may give variable results (inflammation versus lymphoma).
• Clinical signs do not always correlate with severity of histological findings.

Adding to the lack of established histological criteria for grading, and the wide spectrum of
histological findings, is the likelihood that mildly-affected ferrets may not be sampled in the
same way (variable depth and locations of intestinal endoscopic biopsies) as are more
severely affected animals (full thickness samples taken during abdominal surgery)
(P.Cazzini, personal communication). Extrapolation from published diagnostic criteria in
other dogs and cats14 may or may not be directly applicable to ferrets, and currently
immunohistochemistry is often used to separate severe lesions of IBD from lymphoma. A
recent collaborative study (currently in review for publication) is expected to provide data
and insights from a collaborative retrospective study of full-thickness intestinal biopsies from
laparoscopic surgeries15. This study group included an exotic animal clinical resident and
clinician, a clinical pathology resident, and blinded anatomic pathologists from the University
of Georgia, plus one of the authors. They concluded that concurrence between
ferret-experienced pathologists was good, and that immunohistochemical stains were rarely
needed, but that immunohistochemistry may be crucial for a small percentage of animals for
which a diagnosis is uncertain. Otherwise, the lesion may be misdiagnosed.

Common presentations in sugar gliders

Sugar gliders (Petaurus breviceps) are a small marsupial species which are frequently held
by private owners, and they are variably available through pet stores. However, experience
with this species varies between exotic animal specialists, and some may not be familiar
with the common presentations.

Solitary sugar gliders can develop severe skin and tail lesions that are thought to be due to
self-trauma. The lesions appear extremely pruritic and the animals progress to exposing the
underlying musculature. The lesions are severe, focally extensive ulcerative dermatitis and
cellulitis. The overlying crusts may support both coccoid bacteria and yeast16.

Acute to subacute enteritis and colitis are very common lesions with sugar gliders17. These
are usually associated with inflammatory lesions in the liver, heart, kidney and brain.
Specific microbes are seldom recognized, and many cases are suspected to be due to
endotoxemia. A common history is of unexpected death, occasionally with terminal
neurologic clinical signs18.

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

Biliary cystadenomas are fairly common lesions of the hepatobiliary system, seen in older
animals (1.5 to 3 years). These arise most commonly within the liver and less often in
extrahepatic bile ducts. They can be focal or multicentric cystic proliferations, in one or more
hepatic lobes. Focal lesions are generally incidental findings. They are commonly
associated with other liver lesions including hyperplastic bile ducts, lymphocytic cholangitis,
cholangiohepatitis, hepatic lipidosis, and nodular regeneration19.

Sugar gliders (and hedgehogs) are very susceptible to Chagas’ disease, an emerging
pathogen in parts of the USA. American trypanosomiasis (Chagas’ disease) which is caused
by Trypanosoma cruzi, affects dogs, cats, armadillos, monkeys, and small wild animals.
T.cruzi, unlike its relatives, multiplies within the cytoplasm of the mammalian host’s cells. It
has a predilection for cardiac and skeletal muscle where transformation to amastigotes, a
form closely resembling Leishmania sp., occurs within the cells and the collections form
pseudocysts. Infected sugar gliders and African hedgehogs (Atelerix albiventris) die without
significant clinical signs. On post mortem examination, grossly enlarged and thickened
myocardium and hyperemic lungs are common findings. The most significant histologic
lesion is a diffuse lymphoplasmacytic myocarditis with intralesional protozoa that have a
distinct kinetoplast20.

Summary and Future Directions in Exotic Small Mammal Pathology

The ferret study mentioned above, is an excellent example of clinicians, clinical pathologists
and anatomic pathologists working together, building on accepted techniques in domestic
species (dogs and cats) and exploring and validating (or disproving or modifying)
applicability of such techniques to exotic small mammal species. In this and other disease
investigations, pre-planning by diagnostic pathologists can ensure that optimal (and
sometime, extra) sample collection occurs, and that the potential need for ancillary
diagnostic techniques (including clinical pathology, histopathology, immunohistochemical
and PCR tests) are anticipated and satisfied accurately. Key to progress in this area, are the
skills of problem-solving and communication among diagnostic pathologists, to learn from
each other’s experiences, develop new techniques, obtain or develop new antibody or
antigen reagents, and share resources. The authors have found collaboration with wildlife
diagnosticians (especially the Southeastern Wildlife Disease Study Group and the USGS
National Wildlife Health Center) and with U.S. Public Health Service (Centers for Disease
Control) and US Department of Agriculture colleagues to be enormously helpful. Areas for
continued exploration and collaboration include the need for additional validation of test
reagents in various species, and improvement in availability and affordability of small
volume sample collection methods and tests for non-laboratory animals.

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

References:
1. Elliott SP. Rat Bite Fever and Streptobacillus moniliformis. Clinical Microbiology
Reviews, January 2007: 13–22, 2007.
2. Swanson SJ, Snider C, Braden CR, Boxrud D, Wünschmann A, Rudroff JA, Lockett
J, Smith KE. Multidrug-resistant Salmonella enterica serotype typhimurium
associated with pet rodents. N Engl J Med. 356(1):21-8, 2007.
3. Guarner J, Johnson BJ, Paddock CD, Shieh WJ, Goldsmith CS, Reynolds MG,
Damon IK, Regnery RL, Zaki SR; Veterinary Monkeypox Virus Working Group.
Monkeypox transmission and pathogenesis in prairie dogs. Emerg Infect Dis.
10(3):426-431, 2004.
4. Leipig M, Matiasek K, Rinder H et al. Value of histopathology, immunohistochemistry,
and realtime polymerase chain reaction in the confirmatory diagnosis of
Encephalitozoon cuniculi infection in rabbits. J Vet Diag Invest. 25(1):16-26, 2013.
5. Giordano C, Weight A, Vercelli A, Rondena M, Grilli G, Giudice C.
Immunohistochemical identification of Encephalitozoon cuniculi in phacoclastic
uveitis in four rabbits. Vet Ophthalmol. 8(4):271-5, 2005.
6. Graham JE, Garner MM, Reavill DR. Benzimidazole toxicosis in rabbits: 13 cases
(2003 to 2011) J Exotic Pet Med. 23(2):188-195, 2014.
7. Ness RD. Clinical pathology and sample collection in small exotic mammals. Vet Clin
North Am Exot Anim Pract 2(3):591-620, 1999.
8. Pfent CM, Mansell J, Pool RR, Mitchell ME. Pathology in practice. Disseminated
idiopathic myofasciitis. J Am Vet Med Assoc 242(1):43-45, 2013.
9. Eyzaguirre EJ, Walker DH, Zaki SR. Immunohistology of infectious diseases. In:
Dabbs DJ, (Ed.) Diagnostic Immunohistochemistry, Second Edition. Philadelphia:
Elsevier, pp.43-64, 2006.
10. Taylor CR, Shi S-R, Barrr NJ, Wu N. Techniques of immunohistochemistry:
principles, pitfalls and standardization. In: Dabbs DJ (Ed.) Diagnostic
Immunohistochemistry, Second Edition. Philadelphia: Elsevier, pp. 1-42, 2006.
11. Jensen SS, Boenisch T. Antibodies. In: Taylor CR and Rudbeck L (Eds.).Education
Guide: Immunohistochemical Staining Methods, Sixth Edition, Appendix A. Denmark:
Dako, pp.48-60, 2013.
12. Nielsen S. Chapter 4: Selection of the primary antibody. In: Taylor CR and Rudbeck L
(Eds.). Education Guide: Immunohistochemical Staining Methods, Sixth Edition.
Denmark: Dako, pp. 47-60, 2013.
13. Burgess ME. Ferret gastrointestinal and hepatic diseases. In: Lewington JH (Ed.),
Ferret Husbandry, Medicine and Surgery, Second edition. Philadelphia: Elsevier, pp.
203-223, 2007.

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
Reprinted in IVIS with the permission of ACVP Close this window to return to IVIS

14. Day MJ, Bilzer T, Mansell J, et al. Histopathological standards for the diagnosis of
gastrointestinal inflammation in endoscopic biopsy samples from the dog and cat: A
report from the World Small Animal Veterinary Association Gastrointestinal
Standardization Group. J Comp Pathol 138:S1-S43, 2008.
15. Watson K, Cazzini P, Mayer J, Gottdenker N, Reavill DR, Parry N, Fox JG, Sakamoto
K. (in review). Histopathology and immunohistochemistry of severe inflammatory
bowel disease versus lymphoma in the ferret (Mustela putorius furo).
16. Hernandez-Divers SM. Principles of wound management of small mammals:
hedgehogs, prairie dogs and sugar gliders. Vet Clin North Am Exot Anim Pract.
7(1):1-18, 2004.
17. Reavill, DR. Pathology of the exotic companion mammal gastrointestinal system. Vet
Clin North Am Exot Anim Pract 17(2):145-64, 2014.
18. Brust DM. Gastrointestinal Diseases of Marsupials. J Exotic Pet Med. 22(2):132-140,
2013.
19. Reavill D. The pathology of common diseases in small exotic mammals. Presentation
at the Western Veterinary Conference, Las Vegas, NV, February 15-19, 2009.
20. Reavill D. Unusual and unexpected infections: the value of additional testing.
Presentation at the Association of Exotic Mammal Veterinarians Conference
Indianapolis, IN, September 14-19, 2013.

Annual Meeting of the American College of Veterinary Pathologists - 2015 - Minneapolis, MN, USA
View publication stats