Diagnostic Cytology

i n Vet e r i n a r y
Medicine: A
Comparative and
Evidence-Based
A p p ro a c h
a, b
Leslie C. Sharkey, DVM, PhD *, Maxey L. Wellman, DVM, MS, PhD

KEYWORDS
 Cytology  Neoplasia  Infectious disease  Evidence-based
 Veterinary

Cytology is a core diagnostic pathology service that involves specimens from

domestic animals, laboratory animals, and exotic species spanning the phylogenetic
spectrum from invertebrates to marine mammals. Veterinarians in general practice
perform in-clinic cytologic examination of routine lesions such as exudative ears or
subcutaneous masses, which comprise approximately 50% of the samples
collected.1 More complicated lesions usually are evaluated by board-certified veteri-
nary clinical pathologists in veterinary diagnostic laboratories and academic teaching
hospitals, which contributes to increased confidence of the clinician in the diagnosis.1
As the level of sophistication of veterinary medicine has advanced, driven by veteri-
nary medical research, ecological and public health concerns, exponential growth
of veterinary specialists (eg, in oncology, neurology, dermatology), and the human-
animal bond, client expectations for veterinary pathology services also have
increased, resulting in the expansion of veterinary diagnostic laboratories. In
response, training programs in veterinary clinical pathology also have expanded,
with increasing emphasis on diagnostic proficiency and quality assurance.2
Species and breed differences in the prevalence and biology of neoplastic, inflam-
matory, and degenerative diseases in animals present unique and fascinating

Disclosure: Both authors are paid consultants of IDEXX Laboratories.

a
Veterinary Clinical Sciences Department, University of Minnesota College of Veterinary Medi-
cine, 1352 Boyd Avenue, St Paul, MN 55108, USA
b
Department of Veterinary Biosciences, The Ohio State University, 1925 Coffey Road,
Columbus, OH 43210, USA
* Corresponding author.
E-mail address: Shark009@umn.edu

Clin Lab Med 31 (2011) 119

doi:10.1016/j.cll.2010.10.005 labmed.theclinics.com
0272-2712/11/$ see front matter 2011 Elsevier Inc. All rights reserved.
2 Sharkey & Wellman

challenges for veterinary cytopathologists. In this review, the authors share some of
these diagnostic challenges by focusing on selected neoplasms and infectious
diseases that are unique to animals and/or that provide important examples of
comparative disease relevant to humans. The authors hope to emphasize both the
important contributions of medical pathology to veterinary pathology and the impor-
tant role and knowledge base of veterinary clinical pathologists in making cytologic
diagnoses in animals. Where possible, the evidence available for the diagnosis and
classification of these disorders is presented, and areas are identified where additional
research is needed.

EVIDENCE-BASED APPROACH TO VETERINARY CYTOLOGY

In veterinary medicine, as in human medicine, fine-needle aspiration cytology is widely

accepted as an initial diagnostic option for animals with mass lesions, internal organs
with ultrasonographic abnormalities, and other samples, such as effusions. However,
the clinical utility of fine-needle aspirates depends on obtaining a high-quality spec-
imen with adequate cellularity that will be interpreted by an experienced
cytopathologist,3 and ultimately on evidence-based medicine (EBM) that supports
the diagnostic accuracy and clinical relevance of cytology for specific lesions.4
Increased demand for EBM in veterinary cytologybased on clearly defined studies
and comparison with a reference methodhas resulted in part because of rapid
advances in veterinary medical science, newer diagnostic modalities, increased health
care costs for animals, and easier access of clients to electronic media.4 Progress in
veterinary EBM has been impeded by limited funding for controlled studies; the small
numbers of animals usually evaluated; few multi-institutional studies; the variable
impact of species, breed, and geographic location; and obtaining client consent to
enroll their animals in a clinical trial or to collect tissues from their pets for biospecimen
repositories or for research purposes.
Guidelines for evidence-based medical cytology, a relatively new branch of EBM,
often include the need for information about sampling and specimen adequacy,
because these may have a significant impact on interpretation of results.4 For
example, the sensitivity of sputum cytology increases when samples are obtained
over several consecutive days,5,6 and inadequate sampling and improper specimen
handling are major causes of false-negative results in cytologic evaluation of the
cervix.7 Similarly, in dogs, sample cellularity has been shown to be an important deter-
minant of sensitivity and specificity of fine-needle aspirates of bone lesions and
mammary masses in the diagnosis of osteosarcoma and mammary tumors,
respectively.8,9 Criteria to determine the adequacy of fine-needle aspirates include
the numbers of intact cells, cell clusters, and cells within a group; cell types; and blood
or bacterial contamination.4,8,9
Results of cytologic evaluation often are compared with those of histologic evalua-
tion of biopsy specimens to determine sensitivity and specificity, using histopathology
as the reference method.4,8,9 However, assessment of cytologic and histopathologic
specimens by cytopathologists may have a lower interobserver agreement than is
desirable.4 Development of specific cytologic criteria of malignancy for particular
cell types, which are well developed in medical cytopathology but have been
described only for a few animal organs,1013 may improve diagnostic accuracy, as
can the use of ancillary tests such as immunocytochemistry, immunohistochemistry,
and flow cytometry. Various expert computer systems based on Bayesian belief
networks, artificial neural networks, and logistic regression analysis have been devel-
oped for a more objective approach to analysis of cytologic and histopathologic
 Diagnostic Cytology in Veterinary Medicine 3

specimens, such as discrimination between benign and malignant lesions. In veteri-

nary medicine, computerized assessment of mean nuclear morphometry in fine-nee-
dle aspirates has been used to differentiate between benign and malignant mammary
gland tumors and to predict local recurrence of basal cell carcinomas in dogs.1416
A pathology report that clearly and consistently communicates the cytopatholo-
gists interpretation is an important component of evidence-based cytology. Optimum
content and format can be in the form of an open-ended report or checklist.4 Defined
terminology and use of consistent vocabulary to express the probability or likelihood
of cytologic diagnosis may improve communication and the quality of data in cytology
reporting.17 It is important for the clinician to understand the cytopathologists degree
of certainty in the diagnosis of the lesion. Numerical reporting schemes have been
established in human medicine for some lesions,18 but have not been widely applied
in veterinary medicine.
Determination of positive predictive values and other indicators of diagnostic accu-
racy, including correlation with histology and clinical outcome, can improve quality
assurance of cytopathology. In veterinary medicine, correlation between cytologic
and histologic diagnoses depends on the tissue and type of lesion. Correlation is
70% to 90% for skin masses in dogs and cats,13 but only 38% to 60% for splenic
lesions.19 In dogs with neoplastic bone lesions, the correlation between cytologic
and histopathologic diagnoses was 92%, but there was only a 27% correlation for
dogs with nonneoplastic lesions.8 In dogs with benign mammary gland tumors the
correlation between cytologic and histologic diagnoses was 93%, whereas the corre-
lation for dogs with malignant tumors was only 81%.9 Critical evaluation of correlation
studies between cytology and histopathology can be enhanced by using standards for
reporting studies of diagnostic accuracy. Several medical journals have adopted the
Standard for Reporting of Diagnostic Accuracy (STARD), including Veterinary Clinical
Pathology,20 wherein many studies and cases are reported that involve cytology in
animals. Such criteria facilitate complete reporting of methodology and data, optimize
comparison of conclusions with data from similar studies, and ultimately contribute to
the advancement of evidence-based cytology in veterinary medicine.8

DIAGNOSTIC APPLICATION OF CYTOLOGY IN VETERINARY MEDICINE

The advantages of using cytology as a diagnostic modality in animals are similar to

those in humans but often take on an even greater importance in veterinary medicine.
Noninvasive collection methods lower the risk of complications, and rapid availability
of results facilitates triage and timely clinical decisions. Superficial lesions can be
sampled with minimal physical or chemical restraint, compared with biopsy, which
requires local or general anesthesia. Most tissues can be sampled by fine-needle aspi-
ration, and the increased availability of ultrasonography in the veterinary private prac-
tice setting has contributed to increased sampling of internal organs and lesions for
cytologic evaluation. The relatively low cost of routine cytology compared with histo-
pathology, advanced imaging, and other ancillary testing also is a particular benefit in
veterinary medicine, where financial limitations can be a significant consideration
when formulating a diagnostic plan. Unique to veterinary medicine is the fact that cyto-
logic diagnoses can form the basis for advising owners about euthanasia as a humane
option for their pets. In some cases, cytology may be the major or even the sole basis
for such crucial medical decisions, especially if the patient is critically ill or suffering, or
if there are financial considerations that limit additional diagnostic or therapeutic
options. These situations emphasize the importance of high standards for training
and certification of veterinary clinical pathologists, quality assurance guidelines, and
4 Sharkey & Wellman

a comprehensive understanding of the diagnostic sensitivity and specificity of

cytology, which may vary with the type of lesion or pathologic process.21
Cytology often is very useful in differentiating between neoplastic and infectious
processes, and in some cases provides an etiologic diagnosis that is helpful in insti-
tuting a specific chemotherapeutic plan. Most samples for cytologic evaluation in
veterinary medicine are collected by fine-needle aspiration, although impression
smears of biopsy specimens or scrapings of ulcerated or superficial lesions also are
evaluated. Rather than the predominant use of Papanicolaou stain as in medical
pathology, veterinary laboratories and practices typically air-dry samples and use
Romanowsky-type stains for routine evaluation. Papanicolaou staining requires
a multistep procedure and has limitations for evaluating inflammatory reactions,
whereas Romanowsky-type stains like Wright Giemsa stain are rapid, easy to use,
and result in adequate staining of inflammatory and neoplastic cells.22 The differenti-
ation between inflammatory and neoplastic lesions often is a critical step in client
communications and determination of additional diagnostic testing.
Numerous additional diagnostic techniques are used to increase the diagnostic
utility of cytologic specimens from animals. Cytochemical stains such as nonspecific
esterase and alkaline phosphatase have been used to support diagnoses of histiocytic
sarcoma and osteosarcoma, respectively.23,24 Immunocytochemical staining and flow
cytometric analysis are used routinely to determine cell lineage in tumors of hemato-
poietic, epithelial, melanocytic, or mesenchymal origin, and to distinguish between
lymphoma involving B or T lymphocytes, which may have therapeutic and prognostic
relevance in dogs.2536 DNA-based testing such as polymerase chain reaction (PCR)
for antigen receptor rearrangement often is done using fine-needle aspirate speci-
mens to differentiate lymphoid hyperplasia from lymphoid neoplasia, and to determine
whether lymphoid neoplasms are of B- or T-cell origin35,36 (see the article by Bienzle
and Vernau elsewhere in this issue for further exploration of this topic). Although iden-
tifying genetic abnormalities in neoplastic cells from most veterinary species is in its
infancy, this information likely will become important in the future diagnosis and
prevention of disease in animals, especially because breeding options allow the
potential of modifying genetic predisposition for some diseases.37

CYTOLOGIC DIAGNOSIS OF SELECTED NEOPLASMS IN ANIMALS

When establishing a cytologic diagnosis of neoplasia in animals, numerous species

differences in occurrence, prevalence, manifestation, and biologic behavior of the
neoplasms must be considered. Demographic factors such as age, sex and reproduc-
tive status, geography, physical traits such as coat (skin) color, and genetic factors
reflected in breeds or strains within species, add an additional challenge to interpre-
tation. This section describes the cytologic and comparative features of transmissible
tumors, histiocytic neoplasms, melanocytic tumors, and squamous cell carcinoma
affecting different species of animals. These neoplasms are unique to animals or affect
animals in unique ways that contrast with the comparable disease in humans. All of
these neoplasms are diagnosed routinely by veterinary cytopathologists.

Transmissible Tumors
Only two known naturally occurring malignancies can be transmitted by direct engraft-
ment of neoplastic cells between tumor-bearing individuals, and both affect animals:
canine transmissible venereal tumor (TVT) and Tasmanian Devil facial disease (TDFD).
Canine TVT is commonly observed on the face and external genitalia of dogs in
populations where there are large numbers of sexually intact, free-roaming animals.
 Diagnostic Cytology in Veterinary Medicine 5

Cytologic diagnosis is based on identification of a population of round cells with abun-

dant lightly basophilic and highly vacuolated cytoplasm, single round nuclei with
coarse chromatin, and one or two prominent nucleoli (Fig. 1). Lesions may spontane-
ously regress, although chemotherapy is successful for persistent lesions and metas-
tasis has been observed in untreated or immunocompromised dogs. The cell type
involved in TVT remains an unsolved mystery. Genetic analysis suggests the tumor
arose from a single point of origin somewhere between 7800 and 78,000 years ago,
likely from wolves.38 Neoplastic cells have fewer chromosomes (2n 5 5759)
compared with normal canine cells (2n 5 76). This chromosomal abnormality is
uniform in TVTs affecting dogs across the globe,39 the result of gene fusion and rear-
rangement as well as unique genetic mutations in the tumor. Tumor cells express
vimentin and CD45, and are positive for lysozyme and a1-antitrypsin, suggesting
macrophage lineage. During the growth phase of the tumor, major histocompatibility
complex (MHC) class I and II are not highly expressed; however, regression in immu-
nocompetent dogs appears to be associated with increased MHC expression,
possibly triggered by cytokines from infiltrating lymphocytes. Likewise, the tumors
ability to suppress natural killer cells and B cells and to prevent dendritic cell matura-
tion diminishes over time as the tumor succumbs to both cell-mediated and humoral
immune responses in the host, with persistent immunity after tumor regression.
The second transmissible neoplasm, TDFD, is predicted to lead to the imminent
extinction in the wild of its host species, the Tasmanian Devil, a marsupial carnivore
native to the island of Tasmania. In contrast to the long period of coevolution between
TVT and dogs, TDFD was first observed in the mid-1990s. Cytologically, the tumor
cells are large, pleomorphic, and round to spindloid, with large, central, single nuclei.38

Fig. 1. Transmissible venereal tumor from the prepuce of a dog. Note the round-cell
morphology, clear cytoplasmic vacuoles, and moderate anisokaryosis. The cell of origin of
this sexually transmitted neoplasm unique to dogs remains uncertain (Wright Giemsa stain
100 oil immersion).
6 Sharkey & Wellman

The cell of origin has recently been reported to be a Schwann cell.40 The cells are
biologically aggressive in this rapidly progressive and fatal disease, which may be
transmitted by bite wounds. In contrast to TVT, in which more severe disease is
observed in immunocompromised individuals, Tasmanian Devils that succumb to
TDFD are generally immunocompetent. The precise reasons for the difference in
biologic behavior of the two tumors have not been completely elucidated; however,
MHC complexes have low levels of sequence divergence in Tasmanian Devils, which
may allow allograft transmission.

Canine Histiocytic Tumors

Canine cutaneous histiocytoma is a frequently diagnosed round cell tumor that is
unique to dogs. Histiocytomas are single, hairless domed lesions that often occur in
young dogs, with a predilection for the head, ear pinnae, limbs, and trunk, but lesions
may occur anywhere on the body and occasionally are multiple. The diagnosis
frequently is based on the classic clinical presentation and gross appearance, in
combination with characteristic cytologic findings of numerous individual round cells
with moderately abundant basophilic cytoplasm that sometimes contains a few small
clear vacuoles (Fig. 2). Each cell contains a single round, oval, or indented nucleus
with finely stippled chromatin and occasional small nucleoli. Anisocytosis and aniso-
karyosis are mild to moderate. The relatively immature appearance of the cells can
lead cytopathologists unfamiliar with this tumor to be concerned about malignancy,
especially because there often is histopathologic evidence of epidermal invasion,
leading to potential confusion with epidermotropic cutaneous lymphoma. Immunohis-
tochemical staining indicates an epidermal Langerhans cell origin, as the cells are
positive for CD1a, MHC class II, CD11c/CD18, and often cadherin, and negative for
CD4 and Thy-1. Immunohistochemical results are helpful in differentiating canine
cutaneous histiocytoma from other round cell tumors. Canine cutaneous histiocyto-
mas frequently undergo spontaneous remission within a few months of onset, similar
to TVT. Regression often is associated with transient enlargement of the lesion due to

Fig. 2. Canine cutaneous histiocytoma from the forelimb of a dog. These benign neoplasms
usually regress spontaneously, and sometimes are associated with a lymphocytic infiltrate
(Wright Giemsa stain, 50 oil immersion objective).
 Diagnostic Cytology in Veterinary Medicine 7

Fig. 3. Histiocytic sarcoma in a dog. (A) Cytophagic cell in the spleen and (B) cellular pleo-
morphism in the bone marrow (Wright Giemsa stain, 100 oil immersion objective).

massive recruitment of CD81 ab T lymphocytes,41 such that cytologic specimens can

contain large numbers of lymphocytes that in some cases outnumber tumor cells.
Down-regulation of E-cadherin expression also has been documented during
regression.42
Canine cutaneous histiocytoma is one of a group of proliferative histiocytic diseases
that are most common in dogs, infrequently described in cats, and rare or absent in
other animal species. Recent reviews describe the full spectrum of these disorders
in animals.41,43 In contrast to histiocytomas, which are relatively benign lesions, histio-
cytic sarcoma complex (HS) is a rapidly progressive and fatal disease with a significant
breed predilection for Bernese Mountain Dogs, although Rottweilers, flat-coated
retrievers, and Golden retrievers are also predisposed. Primary lesions occur in the
spleen, lymph node, lung, bone marrow, skin and subcutis, and in the periarticular
tissues of the limbs, all sites that are sampled routinely by fine-needle aspiration for
making a cytologic diagnosis.41 Regardless of the site of origin, the cytologic features
include large pleomorphic mononuclear cells with scant to abundant basophilic cyto-
plasm, variable cytophagia, large immature nuclei, multinucleation, and bizarre mitotic
figures (Fig. 3). A hemophagocytic variant of HS exhibits a more diffuse distribution
within affected organs and is often associated with regenerative anemia and thrombo-
cytopenia, which may lead to a temporary misdiagnosis of Evans syndrome.41 Distin-
guishing among HS, other round cell tumors, mesenchymal malignancies, and
non-neoplastic hemophagocytic syndrome in cytologic specimens is augmented by
immunophenotyping: HS cells express CD1, CD11c/CD18, and MHCII, with substitu-
tion of CD11d for CD11c in the hemophagocytic form. HS cells are CD3-negative, dis-
tinguishing them from the large-cell form of T-cell lymphoma, and also typically are
negative for mast cell markers (CD18 variable, CD451, CD45A1, tryptase1, c-kit1).41

Melanocytic Tumors in Horses and Dogs

Melanomas are routinely diagnosed and staged in animals using cytologic methods.
Melanocytic tumors are common in dogs, horses, and some breeds of pigs, less
common in cats and cattle, and rare in sheep and goats.44 Melanocytic lesions are
divided into benign melanocytomas and malignant melanomas. Cutaneous melanocy-
tomas are common in horses older than 5 years with gray or dilute coat color; the
number of tumors increases with age.45 In addition, benign single to confluent lesions
of the deep dermis, concentrated in the perineum, ventral tail, and external genitalia,
can progress over time to multiple malignant tumors in both the skin and internal
8 Sharkey & Wellman

Fig. 4. Metastatic melanoma in fine-needle aspirate specimens of canine lymph nodes. (A)
Highly pigmented melanoma cells and abundant background pigment granules are
admixed with small lymphocytes (Wright Giemsa stain, 50 objective). (B) An amelanotic
melanoma with marked cellular pleomorphism and no apparent melanin pigment. Cell
origin was verified by immunohistochemical staining for S-100 and Melan A (Wright Giemsa
stain, 100 oil immersion objective).

organs. In contrast, horses with nondilute coat color can have benign or malignant
melanomas that are randomly distributed. Recently, a rare equine melanocytic tumor
resembling human intradermal common melanocytic nevi, cellular blue nevi, and
combined cellular blue nevus has been described.45
Certain breeds of dogs have higher (eg, Vizsla, Schnauzer, Chesapeake Bay
retriever) or lower (eg, Siberian husky, Old English Sheepdog, Bichon Frise) statistical
risk of developing melanocytic tumors. In all dogs, benign lesions tend to occur in the
skin and are often black, whereas malignant lesions tend to occur in the oral cavity and
nail bed and have variable pigmentation. Prognosis depends on both the anatomic
location of the lesion and the degree of differentiation.46 Cytologically, well-differenti-
ated melanomas consist of numerous round to polyhedral cells with abundant melanin
pigment and small condensed and uniform nuclei. The less differentiated and more
malignant forms are highly variable cytologically. Malignant melanocytes generally
have marked cellular pleomorphism and can resemble round cells, epithelial cells,
or spindle cells. Pigmentation ranges from heavy to a barely perceptible light-gray
dusting in isolated cells. Nuclei are often large, with marked anisokaryosis, variable
nuclear to cytoplasmic ratios, and large prominent nucleoli (Fig. 4). As in humans,
balloon cell variants of melanoma are rare but have been described in dogs and
cats. These variants are difficult to distinguish cytologically from other cell types
with abundant clear cytoplasm, such as sebaceous cell carcinoma and liposarcoma,
and special stains or ultrastructural studies may be needed for definitive diagnosis.47

Squamous Cell Carcinoma in Animals

Squamous cell carcinomas (SCC) have wide species distribution and are relatively
common tumors in horses, cattle, cats, and dogs. Exposure to solar radiation is
a risk factor in all species, but especially in cattle breeds such as Hereford and
Simmental, which lack pigmentation in the poorly haired circumocular region. Some
breeds of dogs appear to have higher genetic risk for SCC, and white or piebald
dogs with extensive exposure to the sun are also predisposed.44 SCC occurs in the
nail bed of dogs and in the oral cavity of many species, and is the most common tumor
of llamas and horses, where it originates in the squamous epithelium-lined portion of
the stomach.48,49 SCC often is diagnosed cytologically, although well-differentiated
 Diagnostic Cytology in Veterinary Medicine 9

Fig. 5. Squamous cell carcinoma of the pharynx of a dog (Wright Giemsa stain, 50 oil
immersion objective). Cohesive clusters of polygonal to angular cells are visible, some of
which appear to be keratinizing.

tumors may be difficult to distinguish from marked reactive epithelial hyperplasia or

dysplasia, especially when only superficial impression smears are collected and
when there is abundant concurrent inflammation. In the latter cases, histopathology
is required for a definitive diagnosis. Cytologic features of SCC include variably cohe-
sive polyhedral cells in which some or all cells have glassy clear to aqua cytoplasm indi-
cating keratinization; perinuclear vacuolization is noted in some cases (Fig. 5). Nuclei
are round and central, and asynchronous nuclear and cytoplasmic maturation and
a range of immature to well-differentiated squamous epithelial cells often is observed.
Keratinized debris and secondary suppurative inflammation is relatively common, and
there may be secondary bacterial infection or overgrowth of commensal organisms.

CYTOLOGIC DIAGNOSIS OF SELECTED INFECTIOUS DISEASES IN ANIMAL SPECIES

Similar to neoplasms, there are numerous species differences in prevalence, manifes-

tation, and biologic behavior of infectious diseases, and in some cases important
breed differences have been recognized. Etiologic agents that can be recognized in
cytologic specimens include bacteria, fungi, protozoa, rickettsia, and mycoplasma;
viral inclusions; and endo- and ectoparasites. Accurate recognition of infectious
agents is important in diagnosing disease outbreaks in kennels, breeding or research
facilities, herds, and zoos. Diagnosis of zoonotic infections is important in food safety
and security, and in preventing spread of disease to human beings. Although culture,
serology, DNA-based testing, and other sensitive or specific assays can be used to
confirm the diagnosis of most of these infectious diseases, cytology often is the first
diagnostic step and thus can be important in making an initial differential or etiologic
diagnosis. This section describes the cytologic appearance of mycobacteria, the
yeast form of cryptococcosis, the tachyzooites of toxoplasmosis, and the morulae
of erhlichiosis, all of which infect animals and people, with either similar or unique
syndromes associated with infection.

Mycobacteria
Mycobacteria include several groups and individual species of organisms that vary
markedly in host affinity and pathogenicity. In cytologic specimens, these organisms
appear as negatively stained intra- and extracellular rods with Romanowsky stains,
10 Sharkey & Wellman

but are red to magenta when stained with acid-fast stains (Fig. 6). Acid-fast staining of
cytologic specimens from infected tissues can be helpful in making a rapid presump-
tive diagnosis of mycobacteriosis. Mycoplasma infections in animals cause broncho-
pneumonia, pulmonary nodules, and hilar lymphadenopathy, although granulomas in
other tissues and disseminated disease also can occur. Several mycobacteria have
zoonotic potential, and recognition of infection is important for prognosis, treatment,
and prevention (see the article by Carolyn Cray elsewhere in this issue for further
exploration of this topic).
Mycobacterium tuberculosis is a highly pathogenic organism for which human
beings are the only reservoir host.50 Infections in dogs and cats are examples of
anthroponoses because the source, replication, and primary means of transmission
occur in humans; spread of this organism from dogs or cats back to people has not
been reported. Although the prevalence of human and animal infections with M tuber-
culosis has been decreasing in many developed countries, prevalence has been
increasing in underdeveloped countries, and in some countries wildlife has become
endemically infected from close contact with human habitats, serving as reservoirs
for further human infection.50 The majority of reported canine cases of tuberculosis
are caused by M tuberculosis, likely acquired from inhalation of microorganisms in
households shared with tubercular people. The cytology of lesions involving the lungs,
tracheobronchial lymph nodes, and pleura is characterized by a mixed population of
inflammatory cells that includes vacuolated histiocytic cells containing acid-fast
bacilli. The characteristic multinucleated Langerhans cells seen in tubercular lesions
in people are seen less commonly in dogs.51
Infection in people and domestic animals with Mycobacterium bovis is rare, due to
successful eradication programs in ruminants; however, wildlife hosts have become
a reservoir of infection in some areas. Tuberculosis from M bovis is endemic in
white-tailed deer in the northeastern portion of the lower peninsula in Michigan, and
domestic cats have been infected with this organism by ingestion of tissues from
infected deer.52 Calcospherites and granular caseous debris have been described
in a dog with M bovis infection, similar to what has been described in people with
M tuberculosis infection, but have not been described in other types of mycobacterial
infection in dogs and cats.53
Other mycobacterial species often are referred to as atypical mycobacteria. Most of
these organisms are ubiquitous and potentially pathogenic. Mycobacterium avium

Fig. 6. Fine-needle aspirate of a subcutaneous mass from a cat with mycobacteriosis

(Mycobacterium sp). (A) Intracellular nonstaining rods can be seen within macrophages
(Wright Giemsa stain, 100 oil immersion objective). (B) Numerous acid-fast intra- and extra-
cellular organisms appear as magenta rods (Ziehl-Neelsen stain, 50 oil immersion objective).
 Diagnostic Cytology in Veterinary Medicine 11

infections are common in wild, domestic, and captive birds, but are rare in mammals.
Siamese and Abyssinian cats, Basset Hounds, and Miniature Schnauzers may be pre-
disposed to infection with an opportunistic group of mycobacteria called M avium-
Mycobacterium intracellulare.5355 In humans, these mycobacteria usually infect
only immunocompromised individuals. There is no other evidence of spread of these
particular mycobacteria from people to animals or animals to people.50 Cutaneous
lesions characterized by neutrophils, macrophages, lymphocytes, and plasma cells
caused by rapidly growing mycobacteria species such as Mycobacterium fortuitum,
Mycobacterium chelonei, and Mycobacterium smegmatis have been described in
dogs and cats,22 and infection with M fortuitum transmitted by bites from domestic
animals has been reported in people.56 M avium subsp paratuberculosis causes
a chronic inflammatory intestinal disease in cattle called Johnes disease, and has
been implicated as a cause of Crohn disease in people.57
Mycobacteria that infect animals but not people include Mycobacterium lepramae-
murium, which causes murine leprosy, and an unnamed mycobacterium that causes
canine leproid granuloma.58 Feline leprosy syndrome can be caused by several
species of mycobacteria, including M lepramaemurium in cats that become infected
after being bitten by an infected rodent.50 Lymphadenitis and peritonitis caused by
Mycobacterium xenopi infection have been reported in a cat,59 and systemic myco-
bacteriosis due to Mycobacterium marinum complex has been reported in Xenopus
frogs.60 Rapid identification of mycobacteria using cytology is integral to minimizing
zoonotic risk and further spread of disease. In addition to acid-fast stains, other stains
such as auramine O/acridine orange fluorescent staining have been used to detect
mycobacteria. However, none of these stains is as sensitive as immunohistochemical
or PCR analysis in detecting infection.61

Local and Systemic Fungal Infections

The characteristic morphology of some fungal organisms allows them to be recog-
nized in cytologic specimens, although more sensitive and specific assays often are
performed for a definitive diagnosis. Some fungal organisms such as Cryptococcus
are sapronotic, whereby people and animals acquire infections directly from the
environment. Diagnosis of the infection in animals can therefore be important in recog-
nizing contaminated environments and preventing similar exposure and infection in
people. Other fungal infections such as sporotrichosis and dermatophytosis can be
acquired from the environment, or from a scratch or close contact with an infected
animal.
Cryptococcosis occurs in cats, dogs, ferrets, horses, goats, sheep, cattle, dolphins,
birds, koalas, and other marsupials. Most infections occur in dogs and cats, and cats
are more commonly affected than dogs. Infection in dogs and cats most commonly is
due to Cryptococcus neoformans and Cryptococcus gattii, which are likely primary
pathogens.62 Cryptococcus has worldwide distribution and cryptococcosis is an
important fungal disease of immunocompromised human patients, who usually
develop meningoencephalitis. Dogs and cats with cryptococcosis most often present
with clinical signs of nasal cavity infection, although progression to the central nervous
system is not uncommon and gastrointestinal tract infection has been described.63
Koalas may have respiratory tract infections or have asymptomatic colonization of
the nasal passages and skin with C gattii. Subclinical infection and nasal colonization
with C gattii in dogs and cats has been described in evaluation of the recent outbreak
of cryptococcosis in people and animals on Vancouver Island in British Columbia,
Canada.64 Various tree species may be environmental sources for these organisms
in Australia65,66 and Canada.67
12 Sharkey & Wellman

Cryptococcus is a dimorphic fungus that exists in the yeast phase in tissues. The
wide capsule is a critical virulence factor but also is important in recognition of the
organism in cytologic specimens.68 The yeast is round and 4 to 40 mm in diameter,
depending on the width of the capsule, which appears clear with routine Romanowsky
stains. Narrow-based budding of the organism may be apparent and is important for
differentiating Cryptococcus from Blastomyces sp in cytologic specimens (Fig. 7).69
New methylene blue and India ink may be used to enhance visibility of the capsule,22
which may be distorted with routine Romanowsky stains. Poorly encapsulated Cryp-
tococcus strains are more difficult to recognize in cytologic specimens, and may
resemble Sporothrix schenckii or Histoplasma capsulatum. Infection may be associ-
ated with granulomatous or pyogranulomatous inflammation, although there may be
minimal inflammation in some animals. Eosinophilic pleocytosis has been described
with meningoencephalitis caused by C neoformans infection.70 A negative cytologic
examination does not exclude a diagnosis of cryptococcosis. A sensitive and specific
latex agglutination test to detect the capsular antigen in serum is commercially avail-
able, and has been used to monitor response to treatment.70
Sporotrichosis occurs most commonly in dogs and cats, and can be transmitted to
immunocompromised human hosts. Infection is caused by S schenckii, a dimorphic
fungus that occurs as a yeast form in tissues and a telomorph form in soil and on
bushes and trees.71 Infection of both animals and people most often is caused by
skin wounds infected with contaminated soil or plant material, resulting in cutaneous
lesions that can progress to lymphadenitis or occasionally systemic infection. Cats
with sporotrichosis often have multiple draining lesions that typically contain
numerous organisms; these facilitate cytologic diagnosis but can be an important
source of zoonotic infection for people, even in the absence of a skin-penetrating
lesion.71 Transmission from dogs to people is less likely because dogs have fewer
lesions with fewer organisms. Cytologic preparations of lesions caused by S schenckii
are characterized by a mixed cell population of neutrophils, macrophages, lympho-
cytes, and eosinophils, and may contain numerous (cats) or rare (dogs) intra- and
extracellular and intracellular yeasts. The organisms can occur extracellularly or within
neutrophils and macrophages.71,72 With Romanowsky stains, the yeast organisms are
round, oval, or cigar-shaped, 3 to 5 mm wide and 5 to 9 mm long (Fig. 8).69 The

Fig. 7. Cerebrospinal fluid from a cat. A single Cryptococcus neoformans organism with
a wide, nonstaining capsule is surrounded by neutrophils, large mononuclear cells, and
lymphocytes (Wright Giemsa stain, 100 objective).
 Diagnostic Cytology in Veterinary Medicine 13

Fig. 8. Impression smears from an ulcerated lesion on the nose of a domestic shorthair
kitten. Numerous cigar-shaped Sporothrix schenckii organisms can be seen within macro-
phages. Several neutrophils also are present, consistent with pyogranulomatous inflamma-
tion (Wright Giemsa stain, 100 objective).

organism may be surrounded by a clear halo that resembles a capsule. Differential

diagnoses include H capsulatum and poorly encapsulated C neoformans. Definitive
diagnosis is by culture of the exudate from a draining tract or macerated tissue
from a lesion.71,72

Protozoal and Rickettsial Diseases

Toxoplasmosis can affect all warm-blooded animals, including people. Rats, cattle,
horses, and Old World monkeys are relatively resistant to infection, whereas Australian
marsupials and New World monkeys are susceptible to severe and often fatal infec-
tion. Toxoplasmosis is caused by an obligate intracellular coccidian parasite called
Toxoplasma gondii. Infectious oocysts are excreted in feces of domestic cats and
other Felidae, which are definitive hosts.73 Tissue cysts containing the infectious
stages (tachyzoites and bradyzoites) occur in other animals, including people, which
are intermediate hosts.73 Transmission most commonly is congenital, or occurs by
ingestion of cysts in infected tissues (eg, meat), or ingestion of oocysts in contami-
nated food, water, or feces from recently infected cats.73 Tachyzoites can be detected
using routine staining of fine-needle aspirates of various infected tissues and body
fluids, and rarely have been identified in circulating neutrophils and monocytes.74
Infection may be associated with a mixed inflammatory response that includes neutro-
phils and macrophages.75 Tachyzoites are oval, spindloid, or crescent-shaped struc-
tures, 2 to 4 mm long, with light-blue cytoplasm and reddish-purple nuclei that may be
eccentric (Fig. 9).73,75,76 The organisms occur extracellularly or intracellularly within
macrophages or epithelial cells.75 Tachyzoites are similar in morphology to Neospora
caninum and Sarcocystis neurona organisms.74,77 Synchronous fluorescent illumina-
tion or immunocytochemical staining of cytologic samples may be helpful in facilitating
detection of tachyzoites.78 Serologic tests are available, but the results may be difficult
to interpret. PCR assays for DNA in infected tissues is available for definitive
diagnosis.73,74
Ehrlichia organisms are obligate intracellular gram-negative bacteria transmitted by
ticks that feed on infected mammalian wildlife, which may serve as reservoirs for
disease.79 There are several different Ehrlichial organisms with variable geographic
distributions, vectors, wildlife reservoirs, and infected cell types that can cause
14 Sharkey & Wellman

Fig. 9. Impression smear of lung parenchyma from a cat with Toxoplasma gondii infection.
Several crescent-shaped tachyzoites can be seen inside a macrophage (long arrow) and
several extracellular tachyzoites are also present (short arrows) (Wright Giemsa stain,
100 objective).

disease in animals and people. Ehrlichia chaffeensis can infect multiple species79,80
and is considered an emerging infection in people.81 Ehrlichia canis and Ehrlichia
ewengii can infect dogs and people. The small coccoid bacteria occur intracellularly,
in clusters called morulae, which sometimes can be detected in peripheral blood
leukocytes or cells in aspirates of infected tissues. In dogs with E canis, infection
morulae are detected in lymphocytes and monocytes in peripheral blood and lymph
node aspirates from the majority of dogs evaluated.82 In dogs with E ewengii infection,
morulae have been identified in neutrophils, peripheral blood, and synovial fluid.80
With routine stains, morulae are tightly to loosely packed round, purple, mulberry-
like granular structures, one to several micrometers in diameter, within a vacuole
(Fig. 10).7982 Although the cytology of the morulae is characteristic and offers a rapid

Fig. 10. Synovial fluid from a mixed-breed puppy with Ehrlichia ewingii infection. The
neutrophil contains an intracellular morula (Wright Giemsa stain, 100 objective).
 Diagnostic Cytology in Veterinary Medicine 15

diagnosis, few morulae may be present. Definitive diagnosis by serology or PCR is

important because infections are responsive to appropriate antirickettsial agents.

SUMMARY

In this article the authors provide a historical perspective on the development of veter-
inary cytology and brief context in comparison with human cytopathology. Veterinary
cytopathologists must consider several unique entities such as directly transmissible
tumors, as well as extensive species and breed differences in prevalence and diag-
nostic factors, for a variety of neoplastic and infectious diseases common to animals
and humans. Similar to human cytopathology, advances in molecular diagnostics
contribute to increasing the utility of cytology as a sole diagnostic modality. As both
human and veterinary cytopathology evolve, clinicians continue to strive for a rigorous
evidence-based approach to best serve their patients.

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