Nasal Swabs

The presence of an acute or chronic nasal discharge indicates upper respiratory disease but is nonspecific and may be present with inflammatory, infectious, or neoplastic disorders. Nasal discharge may be unilateral or bilateral and range from serous, suppurative, mucoid, to serosanguineous depending on the underlying cause(s). Superficial and deep nasal swabs are easy to obtain and relatively nontraumatic, but often do not provide much information beyond identifying superficial inflammation, secondary bacterial infection, hemorrhage, necrosis, and mucus, while the underlying disease process remains obscure. As a general rule, invasive techniques allowing collection of tissue deep to the nasal mucosa increase diagnostic potential. For example, successful detection of aspergillosis increases from positive detection of 13% to 20% of samples examined by a direct smear or blinds swab to 93% to 100% positive detection in samples obtained by brush cytology or biopsy (De Lorenzi et al., 2006a). However, occasionally the simplest technique can be rewarding, such as the cytologic examination of a nasal swab for the diagnosis of cryptococcosis infection in cats. Therefore, cytologic examination of nasal exudate should be performed initially in any nasal disease.

Nasal Flush

Nasal flushing methods have been reviewed elsewhere (Smallwood and Zenoble, 1993). In general, invasive and aggressive techniques are more likely to yield diagnostic material. Nontraumatic nasal flushes only produce material for a definitive diagnosis in approximately 50% of the cases. A 6 to 10 French polypropylene or soft red rubber urinary catheter is inserted into the external nares to flush sterile, nonbacteriostatic, physiologic saline or lactated Ringer’s solution through the nasal cavity (Fig. 5-1A). A traumatic nasal flush can be accomplished by beveling or nicking the tubing or catheter, creating a rough surface to aid in dislodging tissue. As with any instrument placed into the nasal cavity, penetration through the cribiform plate into the cranial vault can be avoided by measuring the distance from the external nares to the medial canthus of the eye and cutting the tubing or catheter to the appropriate length, or marking the instrument with tape.

FIGURE 5-1 Nasal flush procedure. A, Shown is the placement of a flexible tube within the nasal cavity of an anesthetized dog and use of sodium chloride irrigation fluid. B, Diagram of an alternate technique demonstrating placement of a flexible tube retroflexed below and around the soft palate with collection of fluid from the external nares.

(A, Courtesy of Robert King, University of Florida. B, From Meyer DJ: The management of cytology specimens, Compend Contin Educ Pract Vet 9:10-16, 1987.)

Small aliquots (5 to 10 ml) of fluid are introduced into the nasal cavity via a 20- to 35-ml syringe with alternating positive and negative pressure. As the fluid enters the cavity, the tubing or catheter is aggressively moved back and forth against the nasal turbinates in an attempt to free tissue fragments that can be collected on gauze sponges held below the external nares or reaspirated into the collection syringe. An alternative method involves directing a Foley catheter into the oral cavity and retroflexing around the soft palate into the nasopharynx, inflating the bulb, and lavaging the saline so that the fluid passes through the nasal cavity and out the external nares for collection (Fig. 5-1B).

The fluid and particulate matter retrieved should be placed into an EDTA-anticoagulated tube. If the fluid is turbid, direct smears can be prepared for cytologic evaluation by placing a drop of the fluid on a clean glass slide and placing a second slide on top. After the fluid has spread between the slides, the two slides are pulled apart in a horizontal fashion, with a slight amount of vertical pressure applied if small tissue fragments are present. If the fluid is relatively clear, the sample can be concentrated by centrifugation, and smears are prepared from the sedimented material resuspended in a small volume of remaining supernatant similar to urine sediment preparation. Further concentration of the sample may be achieved via cytocentrifugation, if available. If large tissue chunks are retrieved, touch preparations may be prepared for cytologic evaluation. A small aliquot of fluid can be placed in a tube without any additives for culture and sensitivity, or the fluid may be applied to a culturette.

Fine Needle Aspiration

Fine-needle aspiration (FNA) biopsy is most rewarding when mass lesions are present. If a visible external nasal mass is present, direct aspiration may be performed. To sample masses within the nasal cavity, the location is best identified by imaging techniques prior to aspiration. For FNA, a 1- to 1½-inch 22- to 23-gauge needle is attached to a 3- to 12-ml syringe. The needle is introduced into the mass while strong negative pressure is applied and released several times. The needle should be redirected and the procedure repeated; negative pressure is released before withdrawing the needle from the mass. Frequently, only minimal material is collected into the needle hub. Collected material should be expelled onto slides for cytologic preparation and evaluation.

Imprint and Brush Cytology

Alligator biopsy forceps are used to obtain a pinch biopsy for impression cytology and histopathology, while an endoscopic brush is used to collect tissue to roll on a glass slide for cytology. Both sampling techniques are typically performed with endoscopic guidance. Imprint cytology can also be performed on core biopsy samples obtained using a Tru-Cut Disposable Biopsy Needle (Cardinal Health Allegiance, Deerfield, IL). Similarly, the polypropylene portion of an indwelling catheter with the needle removed, or a polypropylene urinary catheter with the end cut at a 45-degree angle can also be used to obtain tissue specimens. The catheter is pushed into the mass and rotated while applying negative pressure. Tissue can then be rolled on a glass slide or used to make touch imprints for cytologic evaluation before placing in 10% neutral buffered formalin. Brush cytology often misses the deeper inflammatory cells and may not correlate well with histologic results (Michiels et al., 2003). Therefore, deeper, more invasive samples are preferred wherever possible. In one study of 54 dogs with nasal tumors, brush and imprint cytology correctly identified neoplasia of epithelial origin in 88% and 90% of the cases, respectively (Clercx et al., 1996). However, in the same study, the ability to diagnose mesenchymal tumors was significantly less. Histologic diagnosis correlated with 50% of imprint cytology impressions and only 20% of those made by brush cytology.

If the above procedures do not yield diagnostic samples, or cannot be performed because of the nature of the lesion or small patient size, exploratory rhinotomy may be necessary to obtain an excisional biopsy from which impression smears for cytology can be prepared before the remainder of the tissue is preserved for histopathologic examination.

Normal Nasal Cytology

Nasal swabs and flushes of healthy animals contain few cells, small amounts of mucus, and low numbers of a mixed population of extracellular bacteria (normal flora) found colonizing the surface of epithelial cells. Ciliated columnar respiratory epithelial cells from the posterior nasal cavity typically predominate; however, small numbers of squamous epithelial cells originating from the anterior nasal cavity may also be present. Respiratory epithelial cells can be seen singly or in small clusters, are columnar and contain a round, basally located nucleus. Cilia, if present, are located opposite the nucleus and can be seen as an eosinophilic brush border (Fig. 5-2A). While also columnar with a basally located nucleus, goblet cells lack cilia, are more plump, and contain a moderate amount of cytoplasm with numerous prominent, round, purple-staining cytoplasmic mucin granules (Fig. 5-2B). Occasionally, basal epithelial cells may be seen. When present, these cells are round to cuboidal with scant, deeply basophilic cytoplasm and round, centrally placed nuclei. On cytologic specimens, mucus appears as an eosinophilic amorphous extracellular material that often entraps cells. Nasal-associated lymphoid tissue (NALT) can be found in the nasal cavity of both the dog and the cat and responds similarly to organized lymphoid tissue in other location (Fig. 5-3). The canine and feline nasal cavity contains NALT and lymphoid follicles, particularly in the nasopharynx. These islands of lymphocytes can respond similarly to other organized lymphoid tissue such as lymph nodes. The degree of hemorrhage observed is contingent on the collection procedure. Erythrocytes with platelet clumps and white blood cells in numbers and proportions consistent with blood (approximately one white cell per 500 to 1000 red cells) indicate iatrogenic contamination of the sample or peracute hemorrhage. The nasal cavity of the dog and cat harbor large numbers of a mixed population of both aerobic and anaerobic bacteria that are considered normal microflora including Streptococcus sp., Staphylococcus sp., Escherichia coli, Pseudomonas sp., Proteus sp., and Bordetella bronchiseptica.

FIGURE 5-2 Normal nasal epithelium. Tissue aspirate. A, Ciliated columnar epithelium having basally located nuclei is found normally in the upper respiratory tract. (Wright-Giemsa; HP oil.) B, Goblet cells containing large, globular, magenta granules admixed in with ciliated columnar epithelial cells. (Wright-Giemsa; HP oil.)

FIGURE 5-3 Nasal associated lymphoid tissue. Tissue aspirate. Dog. This aspirate contains a heterogeneous mixture of small and medium lymphocytes, lymphoblasts, and increased numbers of plasma cells indicating mild reactive lymphoid hyperplasia. (Wright-Giemsa; HP oil.)

Oropharyngeal Contamination

Oropharyngeal contamination is seen most frequently in samples collected by flushing techniques. The presence of Simonsiella sp. is a hallmark of oropharyngeal contamination. Simonsiella sp. are large, rod-shaped bacteria that align in a row after division resulting in a distinctive pattern that resembles stacked coins (Fig. 5-4A&B). Oropharyngeal contamination is also characterized by the presence of a mixed population of bacteria found extracellularly that colonize the surface of keratinized squamous epithelial cells. If oropharyngeal inflammation is present (e.g., periodontal disease), inflammatory cells may be seen associated with the oropharyngeal contamination (Fig. 5-4C).

FIGURE 5-4 A, Oropharyngeal contamination. TTW. Presence of squamous epithelial cells with closely associated Simonsiella bacteria suggests contamination by normal microflora or an oronasal fistula. (Wright-Giemsa; HP oil.) B, Oropharyngeal contamination. TTW. Degenerating squamous epithelial cell with adherent Simonsiella bacteria and chains of cocci. (Wright-Giemsa; HP oil.) C, Suppurative inflammation with oropharyngeal contamination. BAL. In this case, the source of the inflammation can be difficult to determine. There is obvious suppurative inflammation, and some neutrophils appear to have phagocytized several rod bacteria. However, the presence of squamous epithelial cells with adherent rod bacteria indicates oropharyngeal contamination, which suggests that the inflammation and infection may be localized to the oral cavity. (Wright-Giemsa; HP oil.)

Hyperplasia/Dysplasia

Chronic inflammation secondary to various infectious and noninfectious etiologies (e.g., trauma, chronic irritation, or neoplasia) is common in the nasal cavity and can have a profound effect on the integrity and function of normal cellular constituents. Several adaptive mechanisms are employed by cells to survive amid the inflammatory stimulus. Increased numbers of cells, or hyperplasia, is one such mechanism and is often accompanied by dysplasia (Fig. 5-5A). Dysplasia is readily identified histologically as a loss of architectural organization but is more difficult to identify in cytologic preparations, which typically lack structural features. Samples from an inflamed nasal cavity with hyperplasia and dysplasia are likely to contain numerous clusters and sheets of epithelial cells with an increased nuclear-to-cytoplasm ratio, mild to moderate anisocytosis, and increased cytoplasmic basophilia (Fig. 5-5B). Mitotic figures, while normal in appearance, may be increased as well. Hyperplasia and dysplasia are reversible but may represent early neoplastic changes and can be difficult to differentiate cytologically from well-differentiated carcinoma.

FIGURE 5-5 A, Serous mucous glands of frontal sinus. Tissue imprint. Dog. Clusters of hyperplastic glandular epithelium have an abundant, pale-blue to gray, foamy cytoplasm. (Wright-Giemsa; HP oil.) B, Epithelial dysplasia. Tissue aspirate. This cluster of cells is characterized by increased cytoplasmic basophilia and moderate anisocytosis and anisokaryosis. (Wright-Giemsa; HP oil.)

(A, Courtesy of Rose Raskin, University of Florida.)

Metaplasia

Another adaptive response to chronic irritation/inflammation is metaplasia. Metaplasia involves a change in cellular differentiation such that a susceptible specialized normal cell type is transformed to one that is better able to endure the environmental stress while losing specialized function. In the respiratory system, metaplasia is often characterized by the transformation of columnar respiratory epithelial cells to a more squamous phenotype resulting in a loss of the ability to produce and secrete protective mucus. Cytologically, squamous metaplasia is detected by the presence of squamous epithelial cells either as the primary cell type or admixed with more normal respiratory epithelial cells (French, 1987). Cells may be present in sheets or individually depending on the degree of keratinization. Basilar cells tend to remain in clusters, while more keratinized squamous cells often appear individually and have angular borders, abundant hyalinized, basophilic cytoplasm and small, and occasionally pyknotic or karyorrhectic nuclei. As with hyperplasia, neoplastic transformation of the squamous cells may occur.

Nasal melanosis has also been suggested as a metaplastic transformation of the nasal respiratory mucosa and has been reported rarely in dogs with odontopathic rhinitis (De Lorenzi et al., 2006b).

Foreign Bodies

Nasal foreign bodies occur most commonly in dogs and often originate from plants such as plant awns, foxtails, or twigs. Foreign bodies may be directly inhaled into the nasal cavity, or they may enter the cavity traumatically (e.g., buckshot) through the nares, nasal planum, or via the oral cavity by penetrating the palate. Cytologically, specimens are characterized by marked inflammatory reactions ranging from suppurative to pyogranulomatous often with significant hemorrhage and foreign material such as plant material or fibers. Secondary bacterial infection is common.

Allergic Rhinitis

Hypersensitivity may occur in the nasal cavity alone or concurrent with involvement of the lower airways. The inflammatory infiltrate associated with an allergic rhinitis is characterized predominantly by eosinophils, with lesser numbers of neutrophils, occasional mast cells, and occasional plasma cells (Fig. 5-6). Increased numbers of goblet cells and abundant mucus may also be seen along with rafts of hyperplastic respiratory epithelial cells. Differentials for eosinophilic inflammation include parasitic and fungal infection. Mast cell tumors should also be considered; however, the numbers of mast cells often helps to differentiate these two processes. Typically, mast cells comprise only a small proportion of the inflammatory infiltrate in allergic rhinitis, whereas mast cells tend to be the predominant cell present in mast cell tumors.

FIGURE 5-6 Allergic rhinitis. Nasal flush. Dog. Several eosinophils are enveloped in basophilic mucus that affects the stain quality of the cells. (Wright-Giemsa; HP oil.)

Lymphoplasmacytic Rhinitis

Until recently, only occasional cases of idiopathic lymphoplasmacytic rhinitis had been described (Burgener et al., 1987; Tasker et al., 1999b). This chronic nasal disease in dogs was thought to be immune mediated rather than allergic in origin. However, a more recent study has identified that idiopathic lymphoplasmacytic rhinitis may be more common than previously suspected (Windsor et al., 2004) and may be associated with, and even contribute to, chronic nasal disease in dogs, ultimately resulting in turbinate remodeling and even bony destruction. Despite histologic evidence of bilateral disease in most dogs, a unilateral discharge was seen in some of the cases indicating the need to examine both sides of the nasal cavity even in cases that appear localized in origin. Lack of response to glucocorticoid therapy in the latter report (Windsor et al., 2004) suggests mechanisms other than immune-mediated disease, although multifactorial disease including an immune-mediated component cannot be excluded. Other proposed etiologies include immune dysregulation, allergies, or disruption of the normal microbial flora. Because aspergillosis can also induce a lymphoplasmacytic rhinitis (see Fig. 5-3), there has been concern that idiopathic lymphoplasmacytic rhinitis represents occult aspergillosis. However, analysis of cytokine profiles from nasal biopsies from dogs with these two diseases indicates that the immunologic pattern is quite different. Aspergillosis induces a predominantly T-helper type 1 (Th1) response while a partial Th2 response was detected in cases of idiopathic lymphoplasmacytic rhinitis (Peeters et al., 2007). This type 2 response is in contrast to the type 1 cytokine profile reported in cats with chronic inflammation of the nasal cavity (Johnson et al., 2005), which suggests different underlying etiologies and pathogenesis between these species.

Nasal Polyps

Nasal polyps are occasionally reported in dogs but occur most commonly in cats and are characterized by hyperplasia of the mucous membranes or exuberant proliferation of fibrous connective tissue. Polyps originate within the nasopharyngeal region from the Eustachian tube, middle ear, or nasopharynx. The majority of affected cats are young, often less than 1 year of age (Moore and Ogilvie, 2001). Nasal polyps are speculated to develop secondary to chronic irritation. Although occasional reports have suggested an underlying infectious etiology, the cause of nasal polyps remains unclear in most cases. Inflammatory polyps have the same epithelial and/or connective tissue hyperplasia but also contain a prominent inflammatory infiltrate. Polyps appear grossly as small, smooth, well-circumscribed, pedunculated masses arising from the mucosal surface of the nasal cavity. Clinical signs are usually apparent when the polyp enlarges enough to occlude the nasopharynx. Cytologically, mature lymphocytes and plasma cells are often admixed with rafts of epithelial cells (Fig. 5-7A). Small numbers of neutrophils and macrophages may also be present. Squamous metaplasia and/or dysplasia are frequently seen and, when present, can make the distinction from epithelial neoplasia difficult.

FIGURE 5-7 A, Chronic inflammation. Tissue aspirate. This mononuclear cell population is composed of small and medium-sized lymphocytes and well-differentiated plasma cells. (Wright-Giemsa; HP oil.) B, Chronic rhinitis. Nasal mucosa. Cat. Tissue section demonstrating intact respiratory epithelium with mild to moderate infiltration of mononuclear cells into the lamina propria. (H&E; IP)

(B, Courtesy of Rose Raskin, University of Florida.)

Chronic Sinusitis

Recurrent clinical signs of sneezing and nasal congestion may be related to infectious agents, parasites, allergies, foreign bodies, or neoplasia. A cause may not be demonstrated on cytology or histology in some of these cases. Cytologically, respiratory epithelium appears reactive as evidenced by hyperplasia, dysplasia, or metaplasia. Inflammatory infiltrates often consist of mixed mononuclear cells, including small to medium-sized lymphocytes, plasma cells, and macrophages (Fig. 5-7B).

Bacteria

With the exception of Bordetella bronchiseptica and Pasturella multocida, which may cause acute rhinitis in the dog, primary bacterial rhinitis is rare. However, secondary bacterial infection is common and may accompany nasal neoplasia, viral infection, fungal infection, parasitic infection, trauma, foreign bodies, dental disease, or oronasal fistulation (Fig. 5-8). Infection with Mycoplasma sp. and Chlamydia sp. in cats may cause mild upper respiratory signs concurrently with conjunctivitis.

FIGURE 5-8 Septic suppurative rhinitis. Nasal swab. Cat. Three karyolytic neutrophils are present, one of which has phagocytized Simonsiella sp. bacteria. An active bacterial infection was present in this animal with chronic sneezing and nasal discharge. (Wright-Giemsa; HP oil.)

(Courtesy of Rose Raskin, University of Florida.)

Primary bacterial infection of the nasal cavity is determined cytologically by finding large numbers of a primarily monomorphic bacteria accompanied by a marked suppurative inflammatory response with numerous phagocytized bacteria (Fig. 5-9A). Mucus may be abundant and can obscure identification of bacteria in some cases (Fig. 5-9B&C). Culture of the nasal exudate reveals heavy growth of one type of organism. However, a uniform population of organisms can also be detected with secondary or opportunistic pathogens. Therefore, because bacterial rhinitis is uncommon, significant effort should be made to identify any possible underlying causes. PCR may also be useful for detection of certain organisms, such as Mycoplasma sp. Identification of the bacteria as bacilli or cocci may aid initial institution of antimicrobial therapy as cocci are typically gram-positive and bacilli are typically gram-negative. The presence of filamentous organisms forming mats of colonies suggests Actinomyces and Nocardia spp. Regardless, culture and sensitivity are necessary for proper identification of microorganisms. In addition, whether bacteria are present as a primary or secondary pathogen or as an asymptomatic inhabitant of the nasal cavity, antimicrobial resistance may be of concern.

FIGURE 5-9 A, Bacterial rhinitis. Nasal flush. Large numbers of degenerate neutrophils and a monomorphic population of intracellular and extracellular rod bacteria, consistent with septic suppurative inflammation. (Wright-Giemsa; HP oil.) B, Mucopurulent inflammation. Nasal flush. Degenerate and nondegenerate neutrophils admixed with streams of mucous and nuclear debris. (Wright-Giemsa; HP oil.) C, Septic mucopurulent inflammation. Nasal flush. Closer view of B reveals the presence of intracellular short rod to cocci bacteria that can be difficult to differentiate from the extracellular mucus and cellular debris. (Wright-Giemsa; HP oil.)

Viral

Viral infection of the upper airways often manifests as an acute and transient inflammatory process unless a secondary bacterial infection develops. However, if the viral infection results in turbinate damage and/or epithelial and glandular hyperplasia, chronic rhinitis may follow. Canine distemper virus, adenovirus types 1 and 2, and parainfluenza are the most common etiologies of canine viral rhinitis. Rarely disease may result from infection with herpes virus and reovirus. In cats, feline rhinotracheitis virus (feline herpesvirus I) and feline calicivirus tend to induce moderate to severe upper respiratory signs while reovirus is more often associated with milder symptoms. Severe and recurrent rhinitis is common in cats infected with feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Diagnosis of viral rhinitis is based on patient signalment, history (lack of appropriate vaccination, contact with other animals), clinical signs (mucopurulent nasal discharge, the presence of oral ulcers, conjunctivitis, and fever), direct fluorescent antibody testing of cells obtained from conjunctival scrapings, virus isolation, and/or serology. Cytologic findings associated with viral rhinitis are typically nonspecific with variable numbers and types of inflammatory cells. In addition, the cytology of viral rhinitis is often confounded by the effects of secondary bacterial infection. Viral inclusions are very rarely observed within the epithelial cells.

Fungal

The diagnosis of fungal rhinitis can be complicated because while fungal infection can be a primary disease, fungal organisms may also be found as secondary and opportunistic invaders. In addition, fungi such as Aspergillus sp. and Penicillium sp. can occasionally be cultured from the nasal cavity of clinically normal dogs. Aspergillus sp. and Penicillium sp. are the most common fungal agents in mycotic rhinitis in the dog, whereas Cryptococcus sp. occurs most frequently in cats. Upper respiratory involvement with Histoplasma capsulatum and Blastomyces dermatitidis has also been reported but is rare (Table 5-1).

TABLE 5-1 Mycotic and Protozoal Organisms Commonly Seen in the Respiratory Tract of Dogs and Cats

Aspergillosis and penicilliosis can occur as focal or disseminated respiratory infections in dogs and cats. Both fungi are morphologically similar, necessitating culture for differentiation. Because these two fungi are frequent contaminants of the respiratory tract, diagnosis should be supported by a combination of culture, cytologic, or histologic identification of the organism, and the presence of an inflammatory reaction. German shepherd dogs are frequently afflicted with systemic aspergillosis. While aspergillosis often appears as an opportunistic infection, there are reported cases without obvious predisposing factors.

Infection with Aspergillus sp. can be associated with purulent, granulomatous, or pyogranulomatous inflammation. Infection and inflammation may be present in the nasal cavity alone, the frontal sinus alone, or both sites (Johnson et al., 2006). Cytologically, fungal hyphae are branching, septate, 5-7μm in width, with straight, parallel walls and globose terminal ends. Hyphae can stain either intensely basophilic with a thin, clear outer cell wall or appear as negatively staining images against a cellular background (Fig. 5-10). Hyphae may be difficult to identify when found in low numbers or in dense mats admixed with mucus, inflammatory cells, and cellular debris. Occasionally, round to ovoid blue-green fungal spores may also be observed. Periodic acid-Schiff or silver stains (GMS) are useful when fungal organisms are suspected but not readily identified in the sample. While cytologic characteristics may suggest aspergillosis, culture is necessary for a definitive diagnosis. Similar to bacterial rhinitis, the presence of fungal elements does not rule out underlying neoplasia or other disorders.

FIGURE 5-10 A, Fungal rhinitis. Tissue aspirate. Mat of branching fungal hyphae stain intensely basophilic with prominent septations and globose terminal ends. (Wright-Giemsa; HP oil.) B, Fungal rhinitis. Nasal flush. Dog. Aspergillus fumigatus hyphae shown with degenerate neutrophils is cultured from a secondary infection following treatment for cryptococcosis. (Wright; HP oil.)

(B, Courtesy of Rose Raskin, Purdue University.)

Cryptococcus sp. is a common cause of chronic upper respiratory disease in cats and is also occasionally reported in dogs. However, both Cryptococcus neoformans and Cryptococcus gattii have been reported in the nasal passages of dogs and cats in the absence of local or systemic infection (Duncan et al., 2005; Malik et al., 1997), suggesting that subclinical infection or asymptomatic carriage needs to be considered when the organism is detected in an otherwise normal animal. Inhalation is the suspected route of infection. Concurrent ocular, cutaneous, or neurologic disease may also be seen in animals with Cryptococcus rhinitis. Immunity is speculated to play a role in the development of infections as well as in dissemination of infection throughout the body. Corticosteroid therapy during infection worsens the symptoms as well as the disease progression (Greene, 1998; Medleau and Barsanti, 1990). However, underlying diseases, in particular those that are immunosuppressive (e.g., FeLV, FIV), have not been proven to be predisposing factors to infection (Flatland et al., 1996; Medleau and Barsanti, 1990). Organisms are readily identified in swabs of nasal exudates or imprints/aspirates from nasal masses (Fig. 5-11A&B). Positive identification of the organism via cytology is diagnostic; however, serologic testing and fungal culture may also be useful. New methylene blue (Fig. 5-11C) and India ink can be used to demonstrate the negative staining capsule; however, care must be taken not to mistake air bubbles and fat droplets for organisms. Cryptococcus sp. are round to oval yeast that range in diameter from 8 to 40 μm (including the capsule). The organism has a granular internal structure that stains eosinophilic to purple and is surrounded by a thick, nonstaining, mucoid capsule. The capsule material can give the sample a mucinous texture. Occasionally, narrow-based budding may be seen. Unencapsulated or rough forms are 4 to 8 μm and are difficult to distinguish from H. capsulatum. Fungal culture and serology are useful in this case. The presence and type of inflammation range from the observation of a few to no inflammatory cells amid a field full of organisms to pyogranulomatous inflammation. The degree and type of inflammation may be related to characteristics of the capsule.

FIGURE 5-11 Cryptococcal rhinitis. A, Nasal swab. Numerous yeast forms with distinctive nonstaining, variably thick, mucoid capsules surrounding granular internal structures. The presence of inflammatory cells is variable. (Wright-Giemsa; HP oil.) B, Nasal swab. Narrow-based budding is a feature of Cryptococcus. (Wright-Giemsa; HP oil.) C, Nasal discharge. Cat. Prominent budding and internal structure along with the capsule are highlighted by a water-soluble stain. (New methylene blue; HP oil.)

(C, Courtesy of Rose Raskin, University of Florida.)

Rhinosporidium seeberi occasionally infects the nasal cavity of dogs resulting in single to multiple polyps in which numerous small, miliary sporangia can be observed on the surface. The pathogenesis of infection is unclear, but contact with water and trauma to the nasal mucous membranes are predisposing factors. Cytologically, preparations contain variable numbers of magenta-staining spores that range in diameter from 5 to 15μm. They have slightly refractile capsules and contain numerous, round, eosinophilic structures (spherules). In some cases, the spores stain deeply eosinophilic, preventing visualization of the internal structures. Sporangia are variably sized. They are often very large (range 30 to 300μm) (Fig. 5-12A), well-defined, globoid structures that undergo sporulation to contain numerous small, round endospores (Fig. 5-12B&C). Sporangia are not commonly observed in stained smears because the wall of the sporangia are slightly refractile and do not stain. Sporangia can be observed in unstained direct preparations (Caniatti et al., 1998). Endospores within the sporangia are brown when observed microscopically before staining and appear as three different basophilic forms or stages of maturation with Romanowsky stains (Meier et al., 2006). Immature endospores are approximately 2 to 4 μm in diameter with lightly basophilic cytoplasm and a pink-purple nucleus encompassing ⅓ to ½ of the endospore and 1 to 2 smaller round magenta structures. Intermediate endospores are rarely described but appear to be spherical, granular, basophilic structures approximately 5 to 8 μm in diameter with eosinophilic to globular internal structures and a variably sized, clear halo. Mature endospores tend to predominate in cytologic preparations. These structures are 8 to 15 μm in diameter, with a thick, hyalinized cell wall and a pale, magenta to nonstaining halo. Internal structure can be difficult to visualize in thick areas of the prep, but when endospores are spread out, numerous small spherical eosinophilic globular internal structures can be seen. PAS staining enhances the chance of finding the spores in cytologic or histologic specimens (Fig. 5-12D). Rhinosporidiosis incites a mixed inflammatory response consisting of neutrophils, plasma cells, and lymphocytes. Macrophages, mast cells, and eosinophils are less commonly observed. Rosetting of inflammatory cells, particularly neutrophils, around the spores has been observed and is considered a useful feature in finding the spores during cytologic examination under low magnification (Gori and Scasso, 1994).

FIGURE 5-12 Same case A, C, D. A, Rhinosporidia sporangia. Nasal mass. Dog. Large mature sporangium with numerous endospores expels its contents to the surface (arrowhead). Smaller, variably sized sporangia (arrows) are present within the lamina propria. (H&E; IP.) B, Rhinosporidia endospores. Nasal flush. Dog. Large numbers of round, eosinophilic staining endospores of Rhinosporidium seeberi. Sporangia are rarely seen cytologically. (Wright-Giemsa; HP oil.) C, Rhinosporidia endospores. Tissue imprint. The capsule outline of four endospores is visible along with associated squamous epithelium. (Wright; HP oil.) D, Rhinosporidia endospores. Seven magenta-stained endospores are prominent. (Periodic acid-Schiff; HP oil.)

(A, Glass slide material courtesy of John Bentinck-Smith et al, Mississippi State University; presented at the 1984 ASVCP case review session.)

Sporotrichosis has been rarely identified in samples from the nasal cavity of dogs (Cafarchia et al., 2007; Whittemore and Webb, 2007). The paucity of organisms and cytologic appearance is similar to that reported for Sporothrix schenckii from other canine samples. Sporothrix schenckii has also been isolated in the nasal cavity of cats with sporotrichosis and is more commonly detected in those with cutaneous lesions (Leme et al., 2007).

Nasal mycosis due to infection of cats by Alternaria species, one of the dematiaceous fungi that induce phaeohyphomycosis, has been recently reported in three cats from the United Kingdom (McKay et al., 2001; Tennant et al., 2004). Cytologic findings include the presence of pyogranulomatous inflammation, lymphocytes, and plasma cells. Fungal organisms are pale staining, oval to round with septate hyphae of approximately 7 to 14 μm having a narrow peripheral clear area and finely stippled eosinophilic internal material.

Parasitic

Parasitic rhinitis is uncommon in dogs and cats and may or may not be associated with clinical signs (King et al., 1990). Infection with Eucoleus aerophilus (formerly Capillaria aerophila) is diagnosed by finding the adult nematodes or characteristic ova in nasal secretions. The ova are large (60 × 35 μm), ovoid in shape, with two asymmetrical terminal plugs. Mixed inflammation often containing eosinophils is present. The nasal cavity and frontal sinuses of dogs may be inhabited by several forms of the arthropod parasite Linguatula serrata. Because the ova are infrequently seen in nasal exudates, this parasite is most readily diagnosed by direct visualization via rhinoscopy. The ova measure 90 × 70μm, larvae up to 500 μm, and nymphs measure 4 to 6 mm. Infection with this parasite most commonly elicits mild signs such as sneezing and nasal discharge, but occasionally severe clinical signs occur. The nasal mite Pneumonyssoides caninum is best diagnosed by direct rhinoscopic visualization of the off-white, 1- to 2-mm adult mites inhabiting the nasal cavity and paranasal sinuses of dogs. Infection results in a mild, transient rhinitis.

Protozoal

Leishmania sp. may induce masses in the nasal cavity of dogs. Amastigotes can be identified in aspirates or biopsies from dogs with leishmaniasis (Llanos-Cuentas et al., 1999).

Algal

Prototheca sp. may produce a nasal mass in cats near the nares resulting from a cutaneous infection. Cytologically, aspirate or swab preparations reveal a mixture of inflammatory cells, mostly degenerate neutrophils and macrophages along with numerous sporulated and nonsporulated endospores. The endospores present as variably sized spheres having a thin, clear rim and a granular, dense center (see Fig 3-23A&B).

Epithelial Neoplasia

Malignant epithelial tumors of the nasal cavity occur more frequently than their benign counterparts. The most common epithelial tumors of the nasal cavity include adenocarcinomas, squamous cell carcinomas, transitional carcinomas, and anaplastic or undifferentiated carcinomas. Adenocarcinomas are common in dogs and cats, while SCC are more common in cats (Carswell and Williams, 2007). Cytologic samples from carcinomas tend to be moderately cellular. Neoplastic epithelial cells are present in small aggregates to larger sheets (Fig. 5-13A&B). Adenocarcinomas can be identified by the presence of ring or rosette acinar arrangements that are best visualized at low magnification (e.g., 10×) (Fig. 5-14). Malignant epithelial cells are round to polygonal and typically display numerous criteria of malignancy. Such features include macrocytosis, moderate to marked anisocytosis, anisokaryosis, an increased nuclear-to-cytoplasm ratio, and deeply basophilic cytoplasm that may contain numerous discrete, clear cytoplasmic vacuoles or one large, clear vacuole (signet ring form) suggestive of secretory product. Nucleolar criteria of malignancy should also be assessed, evaluating for the number of nucleoli per nucleus and any size or shape variations. Anaplastic cells may individualize and appear similar to lymphoid cells but large cell size and periodic sheet formation are helpful in distinguishing the two types of neoplasms (Fig. 5-15A&B). Extracellular secretory material such as mucus may also be identified as eosinophilic, amorphous to fibrillar material.

FIGURE 5-13 Nasal carcinoma. A, Nasal flush. Demonstration of cohesive sheets of pleomorphic cells with highly vacuolated cytoplasm. (Wright-Giemsa; HP oil.) B, Nasal aspirate. Cat. Poorly cohesive pleomorphic epithelial cells. Notice variation in cell and nuclear size. (Wright-Giemsa; HP oil.)

FIGURE 5-14 Adenocarcinoma. Tissue imprint. Glandular origin may be identified by the presence of acinar arrangements. (Wright-Giemsa; HP oil.)

FIGURE 5-15 Anaplastic nasal carcinoma. Same case A-B. A, Mass imprint. Dog. Many individualized cells with minimal cohesiveness are present in this highly invasive nasal cavity tumor giving it a “round cell” appearance. (Wright-Giemsa; HP oil.) B, Areas of the slide demonstrate a cohesive, sheetlike epithelial appearance. (Wright-Giemsa; HP oil.)

(A and B, Courtesy of Rose Raskin, University of Florida.)

SCC are distinguished by the presence of cells with angular borders containing abundant, homogenous, glassy cytoplasm and centrally placed nuclei. The neoplastic cells display a wide range in maturation, ranging from immature, small, cuboidal, nucleated, epithelial cells with deeply basophilic cytoplasm to more mature cells, identified as anucleate, fully keratinized cells containing abundant, pale, basophilic cytoplasm and sharply angulated borders. Evidence of asynchronous development may be present such as the identification of fully keratinized cells with retained large nuclei (Fig. 5-16A). Prominent anisokaryosis and variable chromatin patterns ranging from smooth (immature) to clumped (mature) may be seen. A few neoplastic squamous cells may also show a perinuclear clearing (perinuclear “halo”) or even a few, small, clear, punctate, perinuclear vacuoles. Abundant keratinaceous debris represented as amorphous, basophilic extracellular material is often scattered about the slides. A common characteristic of SCC is the presence of a moderate to marked accompanying neutrophilic inflammatory response.

FIGURE 5-16 A, Squamous cell carcinoma. Nasal flush. Asynchronous keratinization, moderate pleomorphism, and perinuclear vacuolation are typical features of squamous cell carcinoma. The associated suppurative inflammation is commonly seen with this type of tumor. (Wright-Giemsa; HP oil.) Same case B-C. Transitional carcinoma. Nasal mass imprint. Dog. B, This appearance is similar to squamous cell carcinoma. Shown is a multinucleate cell with mild neutrophilic inflammation. Notice the pleomorphism of the transitional or nonciliated respiratory epithelium. (Wright-Giemsa; HP oil.) C, Notice the moderately abundant cytoplasm with numerous punctate vacuoles. Malignant features involve anisokaryosis, coarse chromatin clumping, prominent nucleoli, and variable nuclear-to-cytoplasmic ratios (Wright-Giemsa; HP oil.)

(B and C, Courtesy of Rose Raskin, Purdue University.)

Similar in cytologic appearance to SCC is a neoplasm termed transitional carcinoma. This neoplasm arises from nonciliated nasal respiratory epithelium (Carswell and Williams, 2007). It may display a moderately abundant cytoplasm with numerous punctate vacuoles. Malignant features often involve anisokaryosis, multinucleation, coarse chromatin clumping, prominent nucleolus, and variable nuclear-to-cytoplasmic ratios (Fig. 5-16B&C).

Careful documentation of the above characteristics with abundant criteria of malignancy is critical to a diagnosis of neoplasia of the nasal cavity. If criteria of malignancy are not readily apparent, diagnosticians should be cautious as cytologic differentiation of a well-differentiated carcinoma from benign epithelial neoplasia, epithelial hyperplasia, or squamous metaplasia may be impossible, particularly in the presence of inflammation.

Neuroendocrine Carcinoma

Neuroendocrine carcinomas or carcinoids have been rarely described in the nasal cavity of dogs (Patnaik et al., 2002; Sako et al., 2005). Histologically their features appear to be similar to those described elsewhere in the body. Cytologic characteristics have not been reported.

Mesenchymal Neoplasia

Mesenchymal neoplasia of the nasal cavity is uncommon. However, when present, osteosarcoma, fibrosarcoma, and chondrosarcoma are the mesenchymal tumors most often seen (Fig. 5-17). When present, chondrosarcomas are more likely to occur in young dogs with a possible increased risk in medium to large breeds (Lana and Withrow, 2001). Cytologic samples are typically of low cellularity consisting of individualized and occasionally small, loose aggregates of oval, plump, or spindle-shaped cells (Figs. 5-18A&B). Cytoplasmic borders are typically ill defined and neoplastic cells may contain few to moderate numbers of fine eosinophilic to purple cytoplasmic granules. Matrix may be observed as streaming, brightly eosinophilic, fibrillar material often intimately associated with the neoplastic cells. However, this material is easily confused with mucus, and the presence or absence of streaming eosinophilic material on a cytologic preparation should not be used to differentiate the type of neoplasia.

FIGURE 5-17 Frontal sinus sarcoma. Aspirate. Dog. Several individualized oval-to spindle-shaped pleomorphic cells are present in a background of erythrocytes. Several cells contain a faint dusting of azurophilic granules seen in some mesenchymal neoplasias. (Wright-Giemsa; HP oil.)

FIGURE 5-18 Nasal chondrosarcoma. Tissue imprint. Dog. Same case A-B. A, There was a 3-month history of serous nasal discharge and gurgling sounds from nares. Present are pleomorphic individualized cells that display high nuclear-to-cytoplasmic ratios. Several binucleate forms are noted. (Wright-Giemsa; HP oil.) B, Matrix is observed as streaming magenta, fibrillar material intimately associated with the aggregated neoplastic population (arrows). (Wright-Giemsa; HP oil.)

(A, Courtesy of Rose Raskin, University of Florida.)

A cytologic diagnosis of mesenchymal neoplasia is complicated by several factors. Mesenchymal neoplasia often exfoliates poorly resulting in a hemodiluted sample that contains only a few pleomorphic spindle-shaped cells for evaluation. Also, significant inflammation can induce reactive fibroplasia that can be difficult to distinguish from fibrosarcoma. In this case, cytologic evaluation coupled with physical exam and historical and radiographic information raises the index of suspicion for mesenchymal neoplasia, warranting biopsy with histopathologic examination for definitive diagnosis. Additionally, histopathology is often necessary for classification of mesenchymal neoplasia, as the more commonly seen mesenchymal tumors often lack distinguishing cytologic features. Other types of mesenchymal tumors involving the upper airways (see Table 5-2) are uncommon but have cytologic features resembling soft tissue sarcomas in more common sites.

Discrete Cell Neoplasia

Discrete cell (round cell) tumors such as lymphoma, plasmacytomas, mast cell tumors, and transmissible venereal tumors can occur in the nasal cavity. These tumors yield highly cellular preparations composed of individualized, neoplastic, and discrete, round cells with distinct cytoplasmic borders. The morphology resembles that seen in other sites.

Lymphoma is the most common discrete cell tumor reported in the nasal cavity of dogs and cats. In cats, the majority of nasal lymphomas are of B-cell origin, although T-cell lymphoma of the nasal cavity has also been reported (Day et al., 2004; Mukaratirwa et al., 2001). Lymphoma of the nasal cavity tends to be characterized by a monomorphic population of medium-sized or large, immature lymphoblasts with scant, deeply basophilic cytoplasm, large round nuclei, finely granular chromatin, and single to multiple nucleoli (Fig. 5-19). Anaplastic nasal carcinomas (see Fig. 5-15A) can individualize and resemble lymphoma but the presence of very large cells and occasional sheet formation will assist in making the proper diagnosis. Care should be taken to distinguish lymphoma from lymphoid hyperplasia or an inflammatory polyp (see Fig. 5-7A). In lymphoid hyperplasia, a heterogeneous population of lymphocytes and plasma cells are present, with a predominance of small, mature lymphocytes and fewer intermediate-sized lymphocytes and lymphoblasts. In some cases, lymphoma is characterized by a predominance of intermediate-sized lymphocytes with an increased amount of cytoplasm and smooth chromatin lacking nucleoli. Even more problematic are cases where the neoplastic population consists of small, well-differentiated lymphocytes. In such questionable cases, histopathology is imperative for definitive diagnosis of lymphoma.

FIGURE 5-19 Nasal lymphoma. Mass imprint. Cat. Monomorphic population of large round cells with scant cytoplasm, irregularly round nuclei, and single prominent single nucleolus. History included 1-year duration of nasal congestion. (Wright-Giemsa; HP oil.)

(Courtesy of Rose Raskin, University of Florida.)

Canine transmissible venereal tumor (TVT) is a contagious neoplasm involving the external genitalia of both sexes with a low occurrence of metastasis. Spread to the nasal cavity is thought to occur secondary to implantation from a primary genital tumor; however, there are several reports of primary intranasal TVT (Ginel et al., 1995; Papazoglou et al., 2001; Perez et al., 1994). Cytologic preparations reveal large numbers of a monomorphic population of large, round cells with abundant, light to moderately basophilic cytoplasm containing numerous, distinct, small vacuoles. Nuclei are round with coarse to ropy chromatin with 1 or 2 large, prominent nucleoli. Mitoses are frequently observed (Fig. 5-20).

FIGURE 5-20 Transmissible venereal tumor. Nasal mass imprint. Dog. Highly cellular, moderately pleomorphic population of discrete cells with abundant pale cytoplasm, ropy chromatin, and distinct nucleoli. (Wright-Giemsa; HP oil.)

Histiocytic Sarcoma

Canine histiocytic neoplasia can present as either a local or disseminated process. Localized histiocytic sarcomas tend to arise from the subcutis but occasionally originate from other sites including the nasal cavity (Affolter and Moore, 2002). The morphology of cells in this report varied from site to site, as well as within different nodules of the same tumor; however, it was similar in phenotype and variation to those previously described.

Miscellaneous Neoplasia

Oncocytoma of the nasal cavity has been rarely reported in dogs and cats (Doughty et al., 2006) (see section on laryngeal oncocytoma for discussion of cytologic features).

Intranasal and sinus melanoma appears to be uncommon, but has been reported in both dogs (Hicks and Fidel, 2006) and cats (Mukaratirwa et al., 2001).