Guidelines important in evaluating cytological samples for birds

Veterinarians often rely heavily on the results of various diagnostic tests, including hematologic, biochemical, cytologic and immunologic tests to confirm or rule-out the presence of disease and to monitor response to therapy. Of those techniques, cytologic examination of tissues, masses and fluids is often the least expensive, the easiest to perform, and also provides rapid results.

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However, it should also be noted that cytology might not provide a definitive diagnosis. This article will present guidelines for evaluating cytological samples obtained from various sources for avian patients.

Cytology of the conjunctiva and cornea

Cytology samples may be collected from the conjunctiva and cornea using a swab moistened with saline or carefully scraping lesions with a metal or plastic spatula. Local ophthalmic anesthetic agents should be used judiciously when collecting these samples as they are toxic to the cells and may affect the results. Individual epithelial cells or sheets of cells with brown-black pigmented cytoplasmic granules is characteristic of normal conjunctival cytology. These granules should not be confused with bacteria since normal cytology of the conjunctiva may contain a few extracellular bacteria. Inflammatory responses can indicate a bacterial, parasitic, protozoan or mycotic conjunctivitis. Chronic, non-healing corneal lesions should be evaluated for the presence of inflammatory or infectious etiologies and foreign bodies.

Cytology of the skin and subcutaneous tissues

Conventional methods of scraping, tape tests, aspiration, biopsy or tissue imprints may be used to evaluate lesions of the skin and subcutaneous tissues. Skin diseases include, but are not limited to, cutaneous xanthomatosis, feather cysts, neoplasia, inflammation, infections (bacterial, viral, fungal and parasitic diseases) and foreign bodies.

Normal cytology of the skin consists of anucleated cornified squamous epithelial cells, nucleated partially cornified squamous cells, a variable amount of background debris and extracellular bacteria. Mixed-cell populations may indicate an inflammatory or neoplastic condition with secondary infection.

The presence of numerous heterophils and extracellular bacteria is indicative of bacterial infections. Gram's stains are commonly used in avian practice. However, some bacterial pathogens may require special stains other than Gram stains to clearly identify them. Fungal infections are characterized by mixed-cell (macrophages, lymphocytes, plasma cells and giant cells) inflammation with fungal organisms present in the sample. Cutaneous or subcutaneous foreign body lesions often produce mixed inflammatory cell populations consisting of macrophages, multinucleated giant cells and heterophils (See suggested reading). Parasitic infections, such as Knemidocoptic mange, usually are identified by hyperkeratosis of the skin around the cere, feet, legs, eyes or vent and the presence of the mites on skin scrapings. Viral infections, such as avian poxvirus, produce lesions on unfeathered areas of skin around the eyes, cere and feet, as well as lesions in the oropharynx or cornea. Diagnosis of poxvirus infections may be accomplished by scraping the raised lesions and examining them for the presence of large cytoplasmic vacuoles (Bollinger bodies), which contain smaller, round eosinophilic inclusions (Borell) on Wright's stain.

Dermatologic conditions, such as lipomas, xanthomas, lymphoid neoplasia, carcinomas or sarcomas may also be identified by cell type and presence or absence of inflammatory cells. Lipomas often are identified easily following aspiration by the presence of fat droplets and fat cells. Cytology of xanthomas should show macrophagic inflammation with multinucleated giant cells and cholesterol clefts.

Round-cell neoplasms, such as lymphosarcoma, usually yield few individual round cells when aspirated. Carcinomas or neoplasms of epithelial cell origin often yield sheets or clumps of cells of varying size, and nuclear/cytoplasmic ratio characteristic of the type of neoplasia. Neoplasias of mesenchymal cell origin (spindle-cell tumors) also tend to yield few cells that are found as individuals in contrast to epithelial or round-cell tumor types.

Cytology of the digestive tract

Differential diagnoses for plaques, nodules and ulcers in the oropharynx include candidiasis, trichomoniasis, bacterial abscesses, squamous metaplasia due to hypovitaminosis A, papilloma, neoplasia, poxvirus, herpesvirus or physical trauma. These lesions may be assessed by scrapings, swabs, aspiration or imprints of excised tissue. Cytology samples of the esophagus and ingluvies may be obtained using saline-moistened swabs, flushes or washes.

Normal cytology of the oral cavity, esophagus and crop of psittacines can include moderate numbers of Gram-positive bacteria and few to rare Gram-negative bacteria. Candida spp are not commonly present in large numbers except when an infection is present. However, moderate numbers of non-budding yeasts are indicative of a dietary source rather than an active infection. The presence of fungal hyphae indicates a severe fungal infection with possible tissue invasion.

Crop washes are performed by infusing a small amount of saline into the esophagus/ingluvies using a soft plastic or rubber feeding tube, gently massaging the crop and its contents, and aspirating the fluid. The sample may be concentrated by centrifuging the collected material and examining smears of the "pellet."

Cloacal/fecal cytology is often used as a routine part of the physical exam of avian patients. A saline-moistened cotton-tipped swab of appropriate size is gently inserted into the cloaca to obtain a sample. For fecal cytology, fresh is best.

Normal cloacal cytology usually reveals a low to moderate number of squamous cells with varying degrees of keratinization and centrally or eccentrically located vesicular nucleus as well as gram-positive bacteria, an occasional gram-negative or Candida-like yeast and urate crystals The normal fecal bacterial flora of psittacines consists predominately of Lactobacillus spp, Bacillus sp, as well as Staphylococcus and Streptococcus spp. Abnormal samples may contain large numbers of Gram-negative bacteria, many Candida-like yeasts organisms (especially budding ones) and protozoa or parasitic ova. Protozoal organisms, such as Trichomonas sp. or Giardia sp. can be identified using stains specific for those organisms.

Cytology of the respiratory tract

• Infraorbital sinus aspirate/flush: Sinusitis of the nasal and infraorbital sinuses is a condition that often affects psittacine birds. Several methods (with or without anesthesia) may be used to obtain aspirate from the infraorbital sinuses; however, a thorough knowledge of the anatomy of the sinus and surrounding structures is necessary in order to perform these procedures correctly. The first involves restraining the birds head and body and inserting a 20-25 gauge needle (with syringe) at the commissure of the mouth and directed vertically to a point midway between the eye and nares passing under the zygomatic bone. The sinus may be aspirated, or a small amount of sterile saline may be infused into the sinus and then aspirated. The second method involves approaching the sinus at a perpendicular angle and entering the sinus directly. A third method requires entering the sinus from a rostral direction by inserting the needle just caudal to the commissure of the mouth. The needle is directed ventral to the zygomatic arch, ending in the sinus under the eye. Normal cytology of the infraorbital sinus is poorly cellular with little background debris.

• Tracheal wash: In order to properly perform a tracheal wash, the patient should be anesthetized or appropriately restrained; 1-2 ml/kg of sterile saline is infused into the trachea as close to the syrinx as possible and then quickly aspirated. Tracheal aspirations are indicated in patients with clinical signs of a tracheobronchitis (e.g. persistent cough), radiographic evidence of respiratory disease, other evidence of tracheobronchial disease or a lesion involving the syrinx. Normal tracheal cytology is similar to that of the sinuses.

• Air-sac wash: Lower respiratory tract diseases may be diagnosed with the aid of air-sac washes. A small amount of saline (1-3 ml depending upon the size of the patient) may be infused into the appropriate abdominal air sac based upon radiographic evaluation. The caudal abdominal air sac is approached through an aseptically prepared area caudal to the last rib. A small-gauge needle with syringe attached inserted into the air sac, sterile saline is infused and then aspirated. The air-sac wash is accomplished easily with the aid of endoscopy.

Abdominocentesis

Indications for abdominocentesis include ascites, peritonitis, hemoperitoneum or other coelomic cavity fluid accumulation. Abdominocentesis is performed by aseptically preparing a small area on ventral midline caudal to the point of the sternum. A small-gauge needle or butterfly catheter is inserted on ventral midline and directed toward the right side of the coelomic cavity thereby avoiding trauma to the ventriculus and other organs. Any fluid present is aspirated into a sterile syringe. Lavage of the coelomic cavity is performed similarly. Normal abdominal fluid is poorly cellular with an occasional mesothelial cell or macrophage.

Coelomic cavity effusions are classified as pure transudates, modified transudates or exudates (nonseptic, septic, malignant or hemorrhagic) based upon cellularity, color, total protein and specific gravity. Pure transudates resulting from changes in oncotic pressure associated with hypoproteinemias, cardiac disease or hepatic cirrhosis are characterized by a low cellularity (total count less than 1,000 microliters), specific gravity of 1.020 or less, total protein of 3 g/dl or less, and a clear to pale- yellow color. Modified transudates have an increased cellularity (total cell counts greater that 1,000 microliters but less that 5,000 microliters) of mononuclear cells, granulocytes and reactive mesothelial cells, and protein content. Exudates typically have a high cellularity (greater than 5,000 microliters), high specific gravity (greater that 1.020) and total protein levels greater that 3 g/dl. The predominate cell type accompanying exudates may indicate the source.

Cytology of internal organs

Tissue or fluid smears, squash preparations, aspiration or excisional biopsy are methods used to sample internal organs, such as the liver or spleen. Endoscopic or surgical laparoscopy techniques may be used to obtain samples. Splenic impressions generally reveal a significant amount of blood cells and heavy background of cellular debris. Liver aspirates are generally very cellular with hepatocytes in sheets or singles with a large amount of blood cells and free hepatocyte nuclei.

Bone marrow aspiration

Nonregenerative anemias, blood dyscrasias, thrombocytopenia and neoplasia of the hematopoietic and reticulo-endothelial systems should be evaluated by bone-marrow examination. A preferred site for bone marrow aspiration in the avian patient is the proximal tibiotarsal bone. The widest part of the sternum may also be used. Additionally, a particular area may be aspirated if indicated by the presence of a lesion noted on radiographs. The proximal tibiotarsal bone is aseptically prepared, and a small incision is made in the skin. A spinal needle or hypodermic needle is inserted and advanced into the cnemial crest at either the level of insertion of the patellar tendon or perpendicular to the bone on the medial aspect. A 1 to 6-cc syringe is used to collect the sample. The needle and syringe are removed, and the marrow sample is placed on glass slides or cover slips and gently spread. Normal cells present in a bone-marrow aspirate include erythropoietic and granulopoietic cell lines in various stages of development lines, as well as thrombocytes, lymphocytes, osetoblasts and osteoclasts.

Dr. Jones is associate professor of avian and zoological medicine at the University of Tennessee's College of Veterinary Medicine. He is a diplomate of the American Board of Veterinary Practitioners — Avian Specialty. Dr. Jones' clinical interests include raptor medicine, orthopedic and soft-tissue surgery, avian nutrition and avian infectious diseases. He is also a master falconer with 15 years experience.