Susan E. Fielder and Maggie R. McCourt Department of Veterinary Pathobiology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK, USA Clinical signs associated with pathological conditions of the oral cavity include ptyalism, quidding, foul odor, dysphagia, depression, nasal discharge, lymphadenopathy, and weight loss [1]. Conditions of the nasal passages may result in nasal discharge, epistaxis, dyspnea, nasal stertor, reduced air flow, facial distortion, facial swelling, and foul breath. Involvement of the nasopharynx can be associated with dysphagia, dyspnea, abnormal respiratory noise, and exercise intolerance [2]. Horses have seven pairs of paranasal sinuses: frontal, dorsal conchal, middle conchal (ethmoidal), ventral conchal, caudal maxillary, rostral maxillary, and sphenopalatine sinuses. Pathological conditions of the paranasal sinuses may result in unilateral purulent nasal discharge, facial distortion, facial swelling, decreased air flow, foul breath, nasal stertor, dullness on percussion of the involved sinus, and formation of a chronic fistula [2]. The guttural pouches are extensions of the Eustachian tubes that connect the pharynx to the middle ear. Anatomically, they are near the basisphenoid bone, retropharyngeal lymph nodes, pharynx, esophagus, atlantooccipital joint, parotid and mandibular salivary glands, petrous temporal bone, tympanic bulla, and auditory meatus. Clinical signs of guttural pouch disease can be attributed to the specific nerves and arteries associated with the guttural pouch and the auditory system if involved. The internal and external carotid arteries, cranial cervical ganglion, cervical sympathetic trunk and the vagus, glossopharyngeal, hypoglossal, spinal accessory, cranial laryngeal, facial, vestibulocochlear, and mandibular nerves are associated with the guttural pouches. Clinical signs associated with guttural pouch disease include nasal discharge (usually unilateral), unilateral epistaxis, and swelling and/or pain in the area of the parotid salivary gland. The amount of nasal discharge often increases when the head is lowered. Neurological signs (i.e., Horner syndrome) may be present [3]. If disease of the oral cavity is suspected, a thorough examination can usually be conducted in the standing animal with the use of sedation. Food material in the oral cavity may conceal lesions and should be removed by flushing with water or 0.1% chlorhexidine solution before examination. A mouth speculum, headlight, mirror, or oral endoscope may facilitate visualization. Lesions near the base of the tongue are often difficult to visualize and careful digital palpation may be necessary. Radiographic evaluation is sometimes helpful, especially if teeth or bony structures are involved [1]. Diseases of the nasal passages and nasopharynx often require endoscopic examination for adequate visualization. Sedation may distort the nasopharynx by relaxation of the soft tissues. Therefore, if pharyngeal involvement is suspected, initial endoscopic examination of this area should be conducted without the aid of sedation, if possible. Imaging including radiography, computed tomography (CT), or magnetic resonance imaging (MRI) may also help define the extent of lesions in this area [2, 4]. Examination of the paranasal sinuses includes percussion of the sinuses as well as a thorough oral examination. Endoscopy is important in determining the origin of nasal discharge and radiographic evaluation can establish the location and extent of sinus disease. Once sinus involvement is confirmed, the involved sinus can be aspirated for culture and cytological evaluation [2]. The guttural pouches can be evaluated by palpation, endoscopy, and imaging including radiography, CT, and MRI. Endoscopy provides the most information regarding guttural pouch disease and is done under sedation. One method used to enter the guttural pouch is to place a biopsy instrument in the biopsy channel of the endoscope and extend it 2 or 3 cm past the end of the endoscope. Then the biopsy instrument is inserted into the guttural pouch opening and the endoscope rotated to open the guttural pouch flap. Then the endoscope is advanced into the guttural pouch. Another method uses a Chambers mare catheter to rotate and open the flap, allowing the endoscope to enter the guttural pouch (Figure 14.1). The flexible endoscope is passed dorsal or ventral to the catheter and into the pouch as the Chambers catheter is withdrawn [3]. Lesions in the oral cavity, nasal passages, nasopharynx, paranasal sinuses, and guttural pouches may be evaluated by cytological or histopathological examination, and/or culture (bacterial, fungal). Cytological samples from the oral cavity are usually limited to fine needle aspirates of masses or impression smears from excised tissues. Cytological preparations from ulcerative lesions may be collected by imprinting, swabbing, or scraping (see Chapter 12). Figure 14.1 Endoscopic view of the opening of the guttural pouch. A Chambers mare catheter is placed through the opening. Note the yellowish exudate draining from the guttural pouch. Source: Courtesy of Dr C.G. MacAllister. Cytological samples from nasal passages and the nasopharynx are collected directly via the external nares or with a flexible endoscope. Atheromas are accessible for percutaneous aspiration and fungal polyps often are close enough to the external nares to make direct imprints or collect fine needle aspirate or biopsy specimens. Samples of exudates in nasal passages can be collected via polyethylene tubing passed through the biopsy port of a flexible endoscope. Masses and fungal plaques can be sampled with an endoscopic biopsy instrument. Though exudate from paranasal sinuses can be collected endoscopically, samples for cytological evaluation and culture should be taken directly from the involved sinus. Sinus aspiration (sinocentesis) is usually possible in a standing horse using sedation and local anesthesia. After surgical preparation and local anesthetic infiltration, a stab incision is made in the skin and a small Steinmann pin is used to drill into the sinus. Exudate is retrieved by aspiration using an indwelling or rigid catheter. If exudate is not easily obtained, infuse and aspirate 20–30 mL of warm saline. Alternatively, the sinus can be lavaged with 500 mL of warm saline and the nasal discharge examined. If sinus contents are too thick for aspiration, use the eyed end of a large suture needle for sample collection [2]. To collect cytological samples from the guttural pouch, pass polyethylene tubing through the biopsy port of the endoscope and into the guttural pouch. In most instances, exudate is present on the floor of the pouch and is easily aspirated into the tubing. If exudate is not present, infuse 20–30 mL of physiological saline through the tubing and onto the lesion. The saline pools on the floor of the guttural pouch and is easily aspirated. A percutaneous aspiration technique through Viborg’s triangle has been described for guttural pouch lavage and sample collection [5]. Swabbed samples should be gently rolled across a clean glass microscope slide and allowed to air dry. Rolling the swab avoids the rupturing of cells that often occurs if the swab is rubbed or dragged across the slide. Samples collected by brushing should be impressed on the slide. Rubbing or dragging the brush across the slide surface should be avoided to prevent excessive damage to cells. Cells collected by washes can be harvested by centrifugation in a clinical centrifuge using the same speed as is used for urine sedimentation. The supernatant is poured off, the pelleted material is gently resuspended and a drop of the suspension is applied to a clean glass microscope slide with an applicator or pipette. The sediment is then spread using a blood smear technique and allowed to air dry. The oral cavity and upper respiratory tract are composed of several mucous membrane‐lined, communicating passages and cavities: the oral and nasal cavities, pharynx, guttural pouches, and paranasal sinuses. Cytological samples of the normal oral cavity or upper respiratory tract, collected by washing, swabbing, or brushing, consist of the exfoliated epithelial cells characteristic of the area sampled. The epithelium of the mucous membranes lining the oral cavity and rostral portion of the nasal passages consists of keratinized and nonkeratinized stratified squamous epithelium; therefore, these surfaces exfoliate squamous epithelial cells (Figure 14.2). Squamous epithelial cells appear cytologically as large flattened cells with angular borders and abundant, pale‐staining cytoplasm and a condensed to pyknotic central nucleus. Figure 14.2 Swab smear from the oral cavity of a normal horse. Squamous epithelial cells and a multimorphic bacterial population, including Simonsiella, are evident. Source: Courtesy of Oklahoma Veterinary Diagnostics. The nasal epithelium caudal to the vestibule progresses from stratified squamous epithelium to pseudostratified ciliated and nonciliated columnar epithelium with numerous goblet cells and exfoliates these various epithelial cells and goblet cells. Columnar epithelial cells appear cytologically as medium‐sized, elongated cells with basophilic cytoplasm and central to basal nuclei. Ciliated cells have pink‐staining, hair‐like cilia extending as a fringe from one end of the cell (Figure 14.3). Goblet cells contain numerous, red to purple staining cytoplasmic mucin granules (Figure 14.4). The cytological appearance of cells from the oral cavity and upper respiratory tract can be complicated by the presence of cells from specialized structures of the mucosa, such as papillae of various types, and taste and olfactory cells. Figure 14.3 Swab smear from the nasal cavity of a normal horse. Ciliated columnar epithelial cells and goblet cells are evident. Original magnification 250×. Source: Courtesy of Oklahoma Veterinary Diagnostics. Figure 14.4 Goblet cells. Two goblet cells appear as columnar to cuboidal epithelial cells with numerous pink granules in the cytoplasm. Source: Courtesy of Dr R.L. Cowell. Figure 14.5 Swab smear of normal pharyngeal recess. Note cuboidal and columnar epithelial cells and goblet cells. Source: Courtesy of Oklahoma Veterinary Diagnostics. Figure 14.6 Wash sediment from normal guttural pouch. Note cuboidal and columnar epithelial cells and free cilia. Source: Courtesy of Oklahoma Veterinary Diagnostics. The type of cells exfoliated from the pharyngeal mucosa depends on the area sampled. The pharynx is lined primarily by pseudostratified ciliated columnar epithelium, but it also has areas of stratified squamous epithelium (Figure 14.5). The mucosa of the guttural pouches and paranasal sinuses consists of transitional epithelium and simple ciliated columnar or cuboidal epithelium containing goblet cells (Figure 14.6). Cytologically, cuboidal epithelial cells of transitional epithelium appear as medium‐sized, cuboidal cells with rounded borders, basophilic cytoplasm and large central nuclei composed of finely stippled chromatin with areas of condensed chromatin. Bacteria from numerous commensal species normally inhabit the oral cavity and upper respiratory tract in large numbers. This bacterial population is typically heterogeneous and consists of both rods and cocci (see Figure 14.2). Normal bacterial flora do not elicit a significant inflammatory response (i.e., neutrophil exudation) (Figure 14.7). Perhaps the most striking of the normal bacterial flora of the oral cavity are Simonsiella spp., which appear as giant rod‐like structures (see Figure 14.2). These apparent giant rods are composed of multiple Simonsiella rods apposed side to side (Figure 14.8). Figure 14.7 Swab smear from oral cavity. Squamous epithelial cells with commensal bacteria. Source: Courtesy of Dr R.L. Cowell. Figure 14.8 Swab smear from oral cavity. Simonsiella spp. is seen alongside a squamous epithelial cell (bottom). Source: Courtesy of Dr R.L. Cowell. Cytological samples of the oral cavity and upper respiratory tract of horses also often contain “barn mold,” that is, mycelial and fruiting bodies of saprophytic fungi commonly encountered in barn air and feed (Figure 14.9
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Cytology of the Oral and Nasal Cavities, Pharynx, Guttural Pouches, and Paranasal Sinuses
14.1 Indications for Cytological Examination
14.1.1 Oral Cavity, Nasal Cavity, and Paranasal Sinuses
14.1.2 Guttural Pouches
14.2 Examination
14.3 Sample Collection
14.3.1 Oral Cavity
14.3.2 Nasal Passages and Nasopharynx
14.3.3 Paranasal Sinuses
14.3.4 Guttural Pouches
14.4 Sample Preparation
14.5 Normal Cytological Features
14.5.1 Oral Cavity and Nasal Passages
14.5.2 Pharynx, Guttural Pouches, and Paranasal Sinuses
14.5.3 Microorganisms
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