Neoplasia generally is identified by the presence and often the predominance of tissue cells with mild to marked nuclear and cellular pleomorphism (Box 53-1). The more pronounced the magnitude of pleomorphism, the greater is the likelihood of neoplasia and malignancy. Inflammation may be present, particularly if the neoplasm is ulcerated or ruptured. A common dilemma in some cytologic samples is how to differentiate inflammation secondary to neoplasia from tissue dysplasia secondary to inflammation.

Numerous types of neoplasms in all organ systems have been described in reptiles and birds.

8 List the different tumor types and describe general guidelines on their differentiation.

a. Carcinoma: cohesive, round to polygonal epithelial cells, generally with abundant cytoplasm and round to oval nuclei. If the neoplasm is of glandular origin, structures reminiscent of tubules or acini may be present. Carcinomas usually exfoliate well.

b. Sarcoma: elongate-spindle to plump-spindle, discrete mesenchymal cells with variable amounts of cytoplasm and oval to elongate nuclei. Depending on the specific tissue of origin, extracellular matrix may be seen in close association with the neoplastic cells. Sarcomas usually do not exfoliate well.

c. Discrete round cell tumors: discrete, round cells with variable amounts of cytoplasm and round to oval nuclei. Depending on the cell type, cytoplasmic granules may be present (mast cells). These tumors usually exfoliate well.

d. Unknown tissue origin: cytologic characteristics do not readily identify the tissue of origin.

9 What is tissue dysplasia, and how is it identified in cytologic samples?

Tissue dysplasia refers to reversible and atypical morphologic changes in cells that occur in response to inflammation or irritation.

Tissue dysplasia may be identified in cytologic samples in association with variable amounts of inflammation and is cytologically characterized by minimal to moderate nuclear and cytoplasmic pleomorphism (see Box 53-1). The more intense the inflammatory process, the more pronounced may be the dysplastic changes.

10 How does one differentiate between neoplasia with secondary inflammation and inflammation with secondary tissue dysplasia?

Because both dysplasia and neoplasia may share morphologic changes, such as cellular and nuclear pleomorphism, dysplasia secondary to inflammation must be differentiated from neoplasia with secondary inflammation. Typically, when dysplastic cellular changes are caused only by inflammation, the inflammatory process predominates, and the dysplastic changes are not excessive. When neoplasia is present in association with inflammation, the magnitude of the cellular and nuclear pleomorphism is usually greater than the intensity of the inflammatory process. Neoplastic cells usually (but not always) predominate in such samples.

In some cases, cytologic differentiation between dysplasia caused by inflammation and neoplasia may not be possible, and histologic examination is required.

11 What is hyperplasia, and how is it identified in cytologic samples?

Hyperplasia refers to an increase in the numbers of tissue cells in response to a known or unknown stimulus (dependent growth). It is accompanied by hypertrophy, an increase in cell size and cell function.

Hyperplasia may be caused by hormones or local release of inflammatory cytokines, or it may be idiopathic. Hyperplasia may be seen in multiple tissues with or without the presence of inflammation. Hyperplastic cells may exhibit dysplastic changes, particularly when associated with inflammation. Hyperplastic cells without an inflammatory component may be cytologically difficult to differentiate from normal cells. The only clue may be the presence of very high numbers of normal-appearing cells in a sample site that is usually characterized by lower cellularity.

In some cases, cytologic differentiation between well-differentiated neoplasia and hyperplasia may not be possible, and histologic examination is required.

12 What are the cytologic characteristics of inflammation in birds and reptiles?

The hallmark of inflammation is the presence of inflammatory leukocytes in cytologic samples. Heterophils, macrophages, multinucleated giant cells, lymphocytes, plasma cells, eosinophils, and basophils may be present singly or in combination. The proportion of each inflammatory cell type depends on the inciting cause and duration of the lesion.

13 What is the significance of heterophilic inflammation?

Heterophils predominate (>80% of inflammatory cells) in heterophilic inflammation and indicate acute or recent inflammation. They begin to accumulate in tissues within 3 hours of the inflammatory insult. Heterophils are frequently seen in lesions caused by many types of bacteria (with the notable exception of Mycobacterium). Heterophil degeneration may be detected if bacteria (primary or secondary infection) are part of the inflammatory process (Figure 53-1).

Figure 53-1 Aspirate of a subcutaneous mass from a Green iguana depicting intact and degenerate heterophils (arrowheads) with extracellular and intracellular bacteria (arrows). (Wright’s stain; original magnification 1000×.)

14 What is heterophil degeneration?

Heterophil degeneration is characterized by multiple nuclear and cytoplasmic changes that are unrelated to heterophil toxicity. Degenerate heterophil changes are caused by released bacterial toxins at the site of inflammation or infection. These toxins alter the cell and nuclear membrane permeability, primarily causing swelling and vacuolation (Figure 53-2; see also Figure 53-1). Bacteria may be present within heterophils. If degenerate changes are seen, examine the slide carefully for the presence of bacteria within heterophils or in the extracellular space. Degeneration is characterized by the following:

a. Cytoplasmic vacuolation and foaminess

b. Nuclear swelling and chromatin hyalinization (chromatin will appear more eosinophilic and smooth)

c. Karyorrhexis (nuclear fragmentation into numerous, variably sized fragments)

d. Karyolysis (lysis of the nucleus)

Figure 53-2 Impression smears of the epicardial surface from an African Gray parrot hatchling that died of sepsis. Degenerate heterophils (arrows) and numerous extracellular bacteria are shown. Bacterial fibrinous epicarditis was diagnosed by histology. (Wright’s stain; original magnification 1000×.)

Karyorrhexis and karyolysis are generally seen with severe heterophil degeneration.

15 What is the significance of mixed inflammation?

Mixed inflammation refers to an inflammatory process containing a mixture of heterophils (at least 50% of leukocytes), macrophages, lymphocytes, plasma cells, and sometimes, giant multinucleated cells (Figure 53-3). Mixed inflammation is often seen in reptiles and birds. In reptiles, as in birds, mixed inflammation does not necessarily indicate chronicity, since lymphocytes and macrophages may migrate to tissues within a few hours of an inflammatory insult, and multinucleated giant cells form within hours to a few days.

Figure 53-3 Heterophilic granulomatous inflammation in a subcutaneous mass from a Green iguana. A large multinucleated giant cell (center of the image) and multiple heterophils (arrows) are shown. (Diff-Quik; original magnification 500×.)

16 What is the significance of a predominantly macrophagic inflammatory process with or without multinucleated giant cells?

The presence of macrophages and multinucleated giant cells does not necessarily indicate a chronic (several weeks to months) inflammatory process. Conditions in reptiles often associated with a predominantly macrophagic inflammatory process with or without multinucleated giant cells (granulomatous inflammation) include Mycobacterium infection (Figure 53-4), fungal infection, and foreign bodies; xanthomatosis occurs in birds. In contrast to mammals, this type of inflammation may also be seen with many inflammatory conditions, such as bacterial abscesses (birds, reptiles), histomoniasis (turkeys), and chlamydophilosis (birds).

Figure 53-4 Impression smears of the mucosal intestinal surface of an Amazon parrot that died with disseminated mycobacteriosis. Multiple multinucleated giant cells (arrowheads) packed full of numerous nonstaining, rod-shaped mycobacteria (arrow) are shown. (Diff-Quik; original magnification 1000×.)

Granulomatous inflammation is another term often used to describe mixed inflammatory reactions consisting primarily of numerous macrophages with multinucleated giant cells. These reactions may contain variable numbers of lymphocytes and plasma cells.

17 When is the presence of bacteria expected, and when is it an abnormal finding?

a. In general, a heterogeneous population of bacteria is expected in very high numbers from fecal and cloacal cytologic samples and in lower numbers from the oral cavity, crop (birds), and esophagus. Bacteria may be seen on the surface of squamous epithelial cells in tracheal washes due to contamination from the oral cavity.

b. In organs where bacteria are expected (oral cavity, digestive tract), the presence of a monotonous or homogeneous population of bacteria suggests an abnormal overgrowth.

c. In any anatomic location, the presence of inflammatory cells with intracellular bacteria is an abnormal finding and suggests the inflammatory reaction is in response to an active infectious process (primary or secondary infection).

d. The finding of bacteria in organs or tissues that should not contain bacteria (e.g., liver, lungs, air sacs) represents an abnormal finding.

18 How is cytology used in the diagnosis of bacterial infections?

The main advantage of cytology is that, if present in sufficient numbers, bacteria may be quickly and easily visualized (see Figures 53-1, 53-2, and 53-4). A primarily heterophilic or a mixed inflammatory process (with neutrophil predominance) is seen with most bacterial infections, and heterophil degeneration may be an important feature. The inflammatory reaction to mycobacteria consists of macrophages with or without multinucleated giant cells. Mycobacteria do not stain with Romanowsky-type stains and are seen as negative-staining rod-shaped structures within macrophages and giant cells and in the extracellular space (see Figure 53-4). Acid-fast stains are necessary to visualize the organisms.

19 When is the presence of a fungus expected, and when is it an abnormal finding?

Finding fungal organisms (yeasts, hyphae, fruiting bodies) should be considered an abnormal finding in any tissue or organ from a bird or a reptile. In birds, very infrequently seen spherical to oval-shaped yeasts (1 or 2 yeasts per one or two 100×-objective oil immersion fields) in the absence of inflammation may be seen in samples from the upper alimentary tract, feces, and cloaca. Many consider the finding of these low numbers of yeasts in birds to be of no clinical significance.

Greater numbers of yeasts, with or without budding, in the upper alimentary tract, feces, and cloaca are an important finding and indicate yeast overgrowth (Figure 53-5). The presence of hyphae associated with yeasts, with or without budding, in the same anatomic locations mentioned above suggests severe infection with probable mucosal damage and invasion.

Figure 53-5 Fresh fecal smear from an Amazon parrot with yeast overgrowth. Numerous bacteria (arrow) and oval yeasts (arrowhead) are shown. (Diff-Quik; original magnification 1000×.)

20 How is cytology used in the diagnosis of fungal infections?

a. The main advantage of cytology is that, if present in sufficient numbers, fungi may be easily visualized.

b. The inflammatory reaction to fungi in tissue consists primarily of macrophages and multinucleated giant cells (granulomatous inflammation), with variable numbers of heterophils and other inflammatory cells.

c. Fungi may be seen in tissue as yeasts and hyphae. In those species that form hyphae in tissue (e.g., Aspergillus), fruiting bodies may be present and help identify the fungus.

d. The specific morphology of yeasts varies depending on the infecting fungus, but characteristics to look for include the presence of a capsule (Cryptococcus), type of budding (broad vs. narrow base), and size of the yeast.

e. The specific morphology of hyphae vary with the fungus, but characteristics to look for include pigmentation, branching, type of branching (dichotomous vs. nondichotomous), approximate angle of branching (right angles vs. other), hyphae thickness and uniformity (parallel-sided cell walls) and presence or absence of fruiting bodies and other structures (Figure 53-6).

Figure 53-6 Impression smear of caseous material from the oral cavity of a Bearded Dragon lizard. Numerous branching, septate hyphae were seen and are shown in the image. (Wright’s stain; original magnification 1000×.)

21 When is the presence of protozoa expected, and when is it an abnormal finding?

In birds and reptiles, finding protozoal organisms (sporozoites, cysts, trophozoites, merozoites, oocysts) is an abnormal finding in most organs or tissues. However, the presence of some flagellates and ciliates in the stool of reptiles is not always correlated with clinical disease. Infected individuals may serve as carriers and may present with disease if immunosuppressed because of other diseases or conditions.

22 How is cytology used in the diagnosis of protozoal infections?

a. The main advantage of cytology is that, if present in sufficient numbers, protozoal organisms may be easily visualized (Figure 53-7).

b. The inflammatory reaction to protozoa may vary from nonexistent (Giardia in gastrointestinal tract) to heterophilic to a mixed inflammatory process with or without abundant necrotic material.

c. Trichomonas, Sarcocystis, and Giardia organisms are seen in samples from pet and wild birds.

d. Intestinal protozoa other than Giardia are not usually seen in pet birds but are seen in wild birds and poultry.

e. Entamoeba invadens, coccidia, and other protozoa are often seen in reptiles.

f. Morphology of the organisms depends on the specific protozoal agent, but characteristics to look for include number and location of nuclei, flagella, location and number of flagella, shape of organisms, and presence of a cyst wall. With intestinal protozoa it is helpful to determine the combination and number of merozoites and sporocysts per oocyst.

g. Blood protozoa may be encountered during cytologic evaluation of tissues resulting from blood contamination or presence of tissue stages.

Figure 53-7 Three Giardia spp. throphozoites (arrow) in a fresh fecal smear from a budgerigar with loose stools. Giardia throphozoites are characterized by a pyriform shape, two prominent nuclei, and flagella (not shown). (Diff-Quik; original magnification 1000×.)

23 When is the presence of viral inclusions expected, and when is it an abnormal finding?

Viral inclusions (cytoplasmic or nuclear) are an abnormal finding in any tissue of birds or reptiles.

24 How is cytology used in the diagnosis of viral infections in birds and reptiles?

a. Viral inclusions may rarely be seen by cytologic examination of affected tissues.

b. Intracytoplasmic viral inclusions are generally characterized as discrete, single to multiple structures of variable sizes with a homogeneous to finely granular matrix. They may also appear as vacuoles.

c. Intranuclear viral inclusions generally distort and expand nuclei and are identified as hyaline, homogeneous to finely granular material within the nucleus.

d. Viruses that may form cell inclusions in tissue and that affect pet and wild birds include poxvirus, polyomavirus, circovirus, adenovirus, and herpesvirus.

e. Viruses that may form cell inclusions in tissue and that affect reptiles include poxvirus, herpesvirus, adenovirus, inclusion body disease (IBD) virus, and ophidian paramyxovirus.

f. Cytologic diagnosis of poxvirus is easily achieved by identifying the characteristic intracytoplasmic inclusions within affected cells. Although presumptive cytologic diagnosis of viral infections other than poxvirus is possible, diagnosis is most often made using histopathologic examination.

25 How is cytology used in the diagnosis of helminth infections in birds and reptiles?

a. Routine cytologic examination of tissues, masses, and feces may reveal various life stages of helminths. One should keep in mind that the use of cytology is generally not the most sensitive method of diagnosing helminth infections.

b. Adult filarids produce blood microfilariae that may be found in cytologic preparations due to the presence of peripheral blood.

c. Fecal smears, tracheal washes, aspirates, and upper gastrointestinal samples may reveal the presence of helminth eggs or larvae (Figure 53-8).

Figure 53-8 Fecal smear from a salamander infected with Rhabdias spp. lungworms. Two Rhabdias spp. larvae are shown (arrows). Although the sample is not from a reptile, the image demonstrates the benefits of cytologic examination of feces. (Wright’s stain; original magnification 100×.)

26 List some diseases of the avian integument for which cytology may be useful in the diagnosis (Box 53-2).

a. Xanthoma/xanthomatosis

b. Neoplasms

c. Bacterial, viral, fungal and parasitic infections

d. Foreign body reactions

Box 53-2 Avian and Reptilian Integumentary Diseases with Diagnostic Role for Cytology

Xanthoma/xanthomatosis (birds)

Neoplasms: various

Bacterial, viral (poxvirus), fungal, and parasitic infections

Foreign body reactions

27 Describe xanthoma and xanthomatosis.

Xanthomas are nonneoplastic swellings or masses cytologically characterized by accumulations of lipid-filled macrophages, giant cells, and extracellular cholesterol crystals with or without fibrosis. Xanthomas are usually found in the integument but may be found in other locations. The cholesterol crystals are visualized in cytologic preparations as angular, rectangular, negative-staining structures in the extracellular space.

Xanthomatosis is the term used when multiple xanthomas are present. Xanthomatosis is generally associated with increased serum cholesterol and triglycerides and is often seen in birds.

28 What neoplasms of the avian integument can be diagnosed cytologically?

Most neoplasms of the avian integument can be diagnosed cytologically. Although multiple types of tumors have been identified in birds, common neoplasms include the following:

a. Lipomas: often seen in pet birds, particularly budgerigars

b. Epithelial tumors

(1) Squamous cell carcinoma: often seen in fowl, but can occur in many species of birds

(2) Papilloma: may consist primarily of cytologically normal squamous cells

c. Sarcomas

(1) Fibrosarcoma

(2) Liposarcoma

d. Discrete round cell tumors

(1) Lymphosarcoma: frequently seen in fowl with avian leukosis, but may be seen in any species of bird

(2) Melanomas

For detailed cytologic descriptions of these and other tumors, see the Bibliography at the end of this chapter.

29 What bacterial infections of the avian integument can be diagnosed cytologically?

Mycobacterium spp. do not stain with Romanowsky-type stains (see Figure 53-4). They appear as negative-staining structures within macrophages and multinucleated giant cells. Miscellaneous types of bacteria can also be diagnosed cytologically.