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Practical avian necropsy
Practical Avian Necropsy Madeline A. Rae, DVM, MS, DABVP (Avian Practice)
This article is a review of the necropsy techniques utilized in birds with emphasis on psittacine and passerine species. The aim of this article is to assist the practitioner in systematically performing a gross necropsy in the veterinary practice setting and collecting and submitting appropriate samples with the goal of arriving at meaningful diagnoses. Common disease entities are discussed along with the tissues and other samples required for definitive diagnosis of these disorders. 9 2003 Elsevier Inc. All rights reserved. Key words: Avian, necropsy, diagnostics, technique, post-mortem.
he necropsy examination is an i m p o r t a n t part of avian medicine. Often, clinical signs a n d clinical p a t h o l o g i c findings are n o t definitively e x p l a i n e d until necropsy. N e c r o p s y exa m i n a t i o n iS m u c h m o r e t h a n satisfying the curiosity of the owner, b r e e d e r or attending veterinarian. It provides i m p o r t a n t information that can be used in the diagnosis and t r e a t m e n t of future cases. Necropsy findings are an integral part of the flock database f r o m which husbandry, m a n a g e m e n t , treatment, vaccination and quarantine r e c o m m e n d a t i o n s can be made. P o s t m o r t e m information can be invaluable in educating owners of the seriousness of husbandry, nutritional and infectious disease conditions and thereby prevent owners f r o m making the same mistakes in subsequent pets. For the grieving owner, necropsy findings can relieve some or all of the guilt associated with the death of a beloved pet, assure t h e m that they did all they reasonably could, and provide a step in the process toward closure. In situations where the owner may be dissatisfied with t r e a t m e n t or outcomes, it is wise to have a veterinary pathologist p e r f o r m the necropsy. The veterinary patholo-
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From Amazon Veterinary Diagwostics, Salem, OR. Address reprint requests to Madeline A. Rae, DVM, MS, DABVP (Avian Practice), Amazon Veterinary Diagnostics; P.O. Box 7790, Salem, OR 97303-1075. 9 2003 Elsevier Inc. All rights reserved. 1055-937X/03/1202-0002530. 00/0 doi: 10.1053/saep. 2003.127882
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gist should have training, experience and interest in pet and wild birds. T h e necropsy should be p e r f o r m e d as soon after death as is possible. To prevent dry feathers f r o m insulating the body and delaying cooling, wet the feathers with a detergent and water solution. T h e body should be refrigerated, not frozen. Freezing can create artifacts in the tissues that may seriously obscure histologic lesions. P o s t m o r t e m autolysis can also obscure histologic lesions, so if necropsy c a n n o t be perf o r m e d within 3 days, the body should be frozen, realizing that histopathology is likely to be compromised. W h e n shipping a cooled body, be sure the ice-packs (or other frozen coolants) do not directly touch the body, as this may freeze the body tissues, especially in very small birds. Wrap the ice-packs in bubble-wrap or newspaper to prevent freeze damage. Obtain a detailed history, including age of the bird, diet, environment, recent additions to the flock or household, as well as signs of illness. Include any t r e a t m e n t used and if the bird was euthanized, indicate how this was performed. Since intracoelomic injections of barbiturate euthanasia solutions can create extensive lesions, the best euthanasia m e t h o d is to use an overdose of an inhalant anesthetic gas, as this leaves the least a m o u n t of artifactual changes in the body. Even intravenous injections of barbiturates can result in widespread lysis of erythrocytes with pooling of solution in the heart, thereby causing iatrogenic lesions. Instrmnents for the avian necropsy are few. They include a scalpel and handle, thumb-forceps, scissors, a n d rongeur-type i n s t r u m e n t for cutting b o n e and removing the brain. A set of ophthlamic instruments and a h e a d loupe are invaluable in the necropsy of small passerines, neonates or dead-in-shell embryos. Autoclave two sets of the instruments (the rongeurs are only n e e d e d in one set); use one set for o p e n i n g the bird a n d the o t h e r to collect internal organ samples aseptically for culture a n d virus isolation. Microscope slides, coverslips, sealable plastic bags, and formalin bottles or jars should also
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be available. Be sure that formalin fumes do not contact tissues that are to be cultured for bacteria or viruses as this can compromise the culture accuracy. Make sure that formalin fumes do not come in contact with blood or tissue cytological smears as this can severely distort staining and interpretation. Perform the necropsy in a well-lighted, wellventilated area (preferably u n d e r a fume hood) and wear gloves, a mask and if possible a disposable apron. Aerosols from feathers, feces and exudates can be infectious. This is particularly important with cases of chlamydophilosis and mycobacteriosis, which can be zoonotic. However, it is also important to contain the feather d a n d e r and feces in cases of avian polyomavirus and psittacine circovirus infections, so as not to contaminate the premises, your clothing or other adjacent birds. Disinfectant solutions should be readily available for clean-up after the necropsy, but these solutions, nor their fumes, should come in contact with tissues being collected, as they may lyse cells and destroy microorganisms n e e d e d for culture. The particular routine used for gross necropsy of birds can vary, but what remains the same is that all organs are examined. The use of a checklist will ensure that all organs and systems are examined; d o c u m e n t all findings and make this checklist a part of the medical record. It is important to collect samples of everything (all organs, the grossly normal and abnormal). After the necropsy is completed, the decision of which samples to send and what tests to request can be made, but at the very least, the diagnosis does not get cremated with the carcass. Before the necropsy, label sealable bags and formalin jars with the owner's name and the tissues enclosed in an effort to save time and prevent interruptions in the flow of the necropsy.
External Examination The necropsy begins with an external examination. Record the band n u m b e r and scan for microchips; these can be removed, labeled, and saved as p r o o f of identification. Palpate for obvious fractures; in some instances radiographs may be warranted. Examine the skin and feathers. Often feather abnormalities may not be visible while the feather remains in the follicle. For example, the concentric pinching of the feather
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shaft, seen in psittacine circovirus infection, may not be visualized until the feather is plucked from the follicle. Look for stress bars in the wing and tail primaries. Collect multiple blood feathers, both plucked and in the follicle, along with any skin lesions, and place them in formalin. Check for any signs of trauma or bruising. In neonates, closely examine the umbilicus for cleanliness and the adequacy of healing. Examine the unfeathered portions of the legs and the feet for poxvirus lesions, bumblefoot, herpesvirus pododermatitis, and self-mutilation. Examine the uropygial gland, f o u n d at the base of the tail in some species and collect it for histopathologic evaluation, as this can be a site of chronic inflammation and neoplasia. Evaluate the beak, both the external and the intraoral surfaces. O p e n the mouth. Look at and u n d e r the tongue for abnormalities. Look in the choanal slit for mucus and exudate and for blunting of the choanal papillae. Salivary gland enlargement can occur at the base of the tongue and can be due to hypovitaminosis A, bacterial abscesses, or rarely mycobacterial infections. The nares and ear canals should be clear and free of debris or exudate. Examine the conjunctivae and the nictitating membranes. In Columbiformes, these tissues can be collected for Chlamydophila diagnostics, as they may contain elementary bodies. The infra-orbital sinuses should be o p e n e d as aseptically as possible and swabs or aspirates collected for cytology and culture of routine bacteria, Mycoplasma and fungi. Bacterial sinusitis is quite c o m m o n in psittacines, but also occurs in passerine species, and caseous exudate is often seen. In cockatiels (Nymphicus hoUandicus) with 'lockjaw,' sinusitis and temporomandibulitis is c o m m o n , as well as myositis of the mandibular muscles. The mandible and its attached muscles can be placed in formalin for histopathology. In these cases, bacteria such as Bordetella avium, E.nterococcus, E. coli and Enterobacter may be isolated. It is important to indicate to the bacteriology laboratory that Bordetella avium is suspected because this organism is somewhat fastidious and colonies may take longer to appear. Bordetella avium may also cause tracheitis, bronchitis, and p n e u m o n i a in cockatiels and rarely in other psittacines. Several Mycoplasma species have been implicated in conjunctivitis and sinusitis in psittacines and passe-
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rines, but these require special media for isolation and are recovered uncommonly.
Coelomic Cavity The coelomic cavity examination is begun by placing the body in dorsal recumbency, incising the skin of the a b d o m e n and peeling it back caudally over the a b d o m e n and cranially over the pectoral muscle mass. Assess the condition of the pectoral muscle, as this is a good measure of weight loss. Notice whether subcutaneous fat is present or absent and whether there is any bruising or edema. Grasp the sternum with thumb forceps and slightly elevate, maintaining tension on the abdominal skin. Using a scalpel blade, make a transverse incision just caudal to the edge of the sternmn, being careful not to lacerate the liver. Remove the keel and pectoral muscles in one piece by cutting through the ribs and shoulder girdle.
Liver and Spleen Assess the size, color, and consistency of the liver. Hepatic margins should be sharp and not extend beyond the caudal edge of the keel. Note whether a gall bladder is present or absent, as not all species have one. Assess the condition of the airsacs and peritoneal reflections that are visible. If coelomic fluid is present, collect it with a sterile syringe for analysis. Collect samples of airsacs aseptically next, since they are delicate structures that readily disappear with further manipulation of the organs. Aseptically collect liver samples: one sample each for bacteriology, virus isolation or DNA probe testing, Chlamydophila testing, and histopathology. Use any remaining tissue for toxicology a n d / o r impression smears. Grasp the ventriculus, elevate and incise through the attached m e m b r a n e / a i r s a c and then rotate the ventriculus counterclockwise to find the spleen. The spleen is nestled in the cmwe between the proventriculus and the ventriculus. Evaluate the size and shape of the spleen. Determining whether it is of normal size for the bird being necropsied requires some practice, so measuring the diameter can be helpful. The spleen is r o u n d in some species, such as Psittaciformes and Galliformes, and elongated or sock-like in Passeriformes and Columbi-
formes. Note color and any pale foci in the spleen. Collect the entire spleen, dividing it into three samples: one each for virology, Chlamydophila diagnostics, and histopathology. The spleen is the single most important sample for the histopathologic diagnosis o f avian polyomavirus infection, since this is where viral inclusions are most abundant.
Genitourinary Reflect the ventriculus and the intestinal tract to the right side of the bird to view the adrenals, gonads and kidneys leaving the u n o p e n e d gastrointestinal tract for last to avoid contamination of the other abdominal organs. The adrenals are often obscured by active gonadal tissue, so it is easier to collect the cranial division of the kidney with the adrenal and gonad(s) attached for histopathology. Adrenalitis is sometimes noted in unexplained death and may be the only abnormality in some psittacines with proventricular dilation disease (PDD). Sex the bird visually. In most species, only the left ovary and oviduct develop in females, but both testes develop in male birds. The gonads may be pigmented (brown or black) in some species, most notably cockatoos. Note the degree of development of the ovary and oviduct. Is there follicular development? Is so, record the general size of the follicles. Is the oviduct hypertrophied? O p e n the oviduct to look for exudate and tumors, and collect samples for bacteriology and histopathology as needed. Testicular tumors are c o m m o n in budgerigars (Melopsittacus undulatus) and ducks, but seasonal testicular enlargem e n t also occurs and in some species, such as in many passerine birds, can be mistaken for neoplasia; histopathology can usually distinguish between these two changes. The kidneys are nestled in the renal fossae of the synsacrum, with the lumbosacral plexus lying deep to the caudal division of the kidney. The ureters run down the ventral surface of the kidneys bilaterally. In addition to the kidney/adren a l / g o n a d tissue collected above for histopathology, aseptically collect additional renal samples for virology, toxicology, and bacteriology (if exudate is present). In small birds ( u n d e r 30 g), one can make an en bloc excision of the kidneys still in situ within the synsacrum and place this in formalin. After fixation, renal dissection is easier
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metabolic b o n e disease needs to be considered. Parathyroid h y p e r t r o p h y is usually the only gross pathologic lesion f o u n d in the hypocalcemia syndrome of African gray parrots (Psittacus er-
ithacus)
Cardiac
Figure 1. Anatomic location of normal avian thyroids with the keel removed. Thyroids (a) are located on either side of the trachea. The normal parathyroids are barely visible as they are usually closely associated with the caudal poles of the thyroids. Trachea (b). Carotid arteries (c). Heart (d). a n d / o r the synsacrum can be decalcified a n d cross-sections of kidney together with b o n e can be cut. After removal of the kidneys, evaluate the lumbosacral plexus, especially in cases of pelvic limb weakness or malfunction; samples of these nerves can be collected in formalin for histopathologic evaluation.
Thoracic Inlet Move to the thoracic inlet region. Identify the thyroids a n d parathyroids, located cranial to the heart and adjacent to the carotid arteries bilaterally, a n d collect t h e m for histopathology. (Fig 1) Goiterous changes were once quite c o m m o n in budgerigars, but are less so with the advent of c o m m e r c i a l diets. Hyperplastic goiter has b e e n seen in juvenile macaws recently. Lymphocytic thyroiditis may also be seen histologically, especially in A m a z o n parrots. N o r m a l parathyroids are barely visible. W h e n they are p r o m i n e n t ,
Examine the heart, p e r i c a r d i u m and great vessels. Visceral gout can cause the deposition of white, m u c o i d urate material on the pericardial sac. View a s m e a r of this material u n d e r polarized light to confirm the presence of uric acid crystals. Formalin fixation may dissolve uric acid crystals a n d they may not be visible on histopathology (crystals do not dissolve if fixed in ethanol, but this is usually not a practical fixative for other reasons). Suppurative pericarditis can be caused by a variety of bacteria, such as Pasteurella and Chlamydophila. Cytologic examination of the pericardial exudate may reveal the causative organisms. H y d r o p e r i c a r d i u m is a c o m m o n finding in avian polyomavirus infection in juvenile psittacines. Before removing the heart f r o m the thoracic cavity, heart b l o o d can be collected using a sterile syringe a n d needle for bacteriology. Smears of heart b l o o d can be stained with Wright's stain and e x a m i n e d for h e m o p r o t o z o a and microfilaria, or G r a m stained to look for bacteria. After removing the heart and great vessels, o p e n the heart in the direction of blood flow, using water to rinse away b l o o d and clots. L o o k for thrombi, valvular endocarditis lesions, a n d pale areas in the myocardium. Congenital cardiac anomalies are rare. O p e n the great vessels to look for atherosclerosis, which may involve the aorta, p u l m o n a r y artery or carotids. Atherosclerosis is characterized grossly by yellowish, raised, intimal plaques, but may occasionally be so severe that the carotids are completely obstructed. Mineralization of the great vessels may also occur in association with atherosclerosis or related to renal disease and hypervitaminosis D. Atherosclerosis is most c o m m o n l y seen in African gray parrots, where it can be mild to moderate, but also in obese, older Amazon parrots (Amazona spp.) a n d captive raptots, where it can be so severe that it results in acute death. Atherosclerotic lesions may also be f o u n d in the coronary arteries, but usually these
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lesions are discovered on histopathologic examination. It is best to place most of the heart in formalin, so that multiple sections can be taken for histopathologic evaluation. Pale loci or streaks can indicate degenerative m y o p a t h y related to vitamin E / s e l e n i u m deficiency or myocarditis associated with septicemia or viral diseases, such as West Nile virus or PDD. Petechial and ecchymotic epicardial h e m o r r h a g e s are c o m m o n l y seen in cases of acute death f r o m avian polyomavirus.
Respiratory Examine the lungs in situ before removing them. Avian lungs are fixed in place within the avian thoracic cavity, and are not freely moveable. Removal requires gentle teasing of the lung tissue away f r o m the ribs. The avian lung is one tissue in which gross lesions may a p p e a r quite significant, but on histopathologic evaluation turn out to be just passive congestion. Conversely, grossly n o r m a l lungs may contain significant histologic lesions. So, it is wise to always include lung for histopathology, even if it appears grossly normal. Collect a portion of the lung for bacteriology and virology and place the rest in formalin for histopathology. Because lesions can be focal or multifocal, it is best to include a large portion of at least one lung. In canaries and mynahs, a Wright's-stained impression s m e a r of lung (along with impressions from liver and spleen) is very i m p o r t a n t in detecting the monocytic f o r m of atoxoplasmosis, as Atoxoplasma organisms are not visible on histopathology. Sarcocystis organisms can also be seen in impression smears of the lung.
Oral Cavity and Gastrointestinal Tract Cutting t h r o u g h the mandible at one of the lateral commissures allows access to the caudal p h a r y n x and visualization of the glottis. T h e larynx and trachea can then be o p e n e d down to the tracheal bifurcation, looking for hemorrhage, exudate, foreign bodies, granulomas, and parasites, such as respiratory mites or Syngamus nematodes. Tissue samples should be collected for histopathology and virus isolation, as well as
bacteriology and fungal culture if warranted. Fungal tracheitis, especially at the syrinx, can be diagnosed by cytologic examination of exudate or g r a n u l o m a t o u s material, fungal culture, a n d / o r by histopathology. Rarely, syringeal granulomas can be caused by mycobacterial organisms. In canaries, Enterococcus faecalis can cause chronic tracheobronchitis; culture of the tracheal l u m e n is necessary for diagnosis. Viral tracheitis is rare in psittacine birds, but a herpesviral tracheitis, bronchitis and airsacculitis has b e e n r e p o r t e d in Neophema parrots. Canarypox can p r o d u c e a severe tracheobronchitis with intracytoplasmic inclusions. Going back to the pharynx, the cut can extend downward the length of the esophagus and into the crop, looking for lacerations a n d punctures, peri-esophageal abscesses, and o t h e r abnormalities. In game birds, the esophagus and crop may exhibit m o d e r a t e to m a r k e d thickening and m u c u s p r o d u c t i o n due to capillariasis. A wet m o u n t scraping f r o m the crop can reveal the typical bipolar capillarid ova. In juvenile psittacines, thickening and 'Turkish towel' app e a r a n c e of the crop mucosa is often due to candidiasis and either a wet m o u n t smear, cytology or G r a m stain of a crop mucosal scraping can be diagnostic. T r i c h o m o n a d s can be f o u n d in wet m o u n t s f r o m the oral cavity a n d / o r crop of Columbiformes and raptors, but may also occasionally b e f o u n d in the crops of budgerigars and passerines. T h e crop contents can be collected in a plastic bag and frozen, if there is any suggestion o f a toxin ingestion. A large section of crop, to include a big vessel and adjacent nerve, should be collected for histopathology, since PDD lesions are often confined to the nerves. At this point, the esophagus distal to the crop can be transected. Caudal traction of the distal esophagus and sharp dissection of the mesenteric attachments can be used to remove the entire gastro-intestinal tract. Continue the dissection to m a k e a circular incision a r o u n d the vent, leaving a margin of intact vent skin and the bursa of Fabricius attached to the tract. The bursa is present in young birds, usually less than 6 to 12 m o n t h s o f age, and is located dorsal to the cloaca. (Fig 2) The bursa should always be collected w h e n it is present and divided in hale Submit one half in formalin for histopathology a n d save the other half for virology a n d / o r DNA
Practical Avian Necropsy
Figure 2. Anatomic location of the normal avian bursa in a 4-month-old Amazon parrot (Amazona sp). The bursa (a) is located dorsal to the cloaca. The colon (b) is retracted caudally to expose the bursa. The caudal division of the kidney is noted (c).
p r o b e testing. T h e bursa is i m p o r t a n t in diagnosing psittacine circovirus, especially in young African gray parrots that die acutely without feather lesions, since the bursa may be the only site where viral inclusions are found. Lesions in the bursa are often nonspecific as to etiology, but can indicate the acuteness or chronicity of stress. O p e n the distal esophagus with scissors, continuing on into the proventriculus and ventriculus. Evaluate the stomach contents for a m o u n t and foreign material. Collect and freeze the contents for possible toxicologic analysis. Rinse the mucosa with water and m a k e wet m o u n t and dried smears of m u c u s a n d / o r mucosal scrapings. Do not separate the proventriculus and ventriculus. T h e isthmus (the junction between the proventriculus and ventriculus) is a c o m m o n site for avian gastric yeast (formerly known as megabacteria; suggested new name, Virgamyces avigast'r and gastric carcinoma. Collect a large specimen of proventriculus, isthmus, and ventriculus (all in one piece, if possible), con-
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taining at least one large serosal nerve and blood vessel, for histopathology. A large specimen allows multiple sections to be e x a m i n e d by the veterinary pathologist in the search for nerves a n d plexi. Dilation of the proventriculus a n d / o r ventriculus is a hallmark gross lesion of PDD, but in juvenile psittacines being hand-fed, these organs may also be dilated as a n o r m a l finding. Histopathology is required to differentiate between PDD and n o r m a l juvenile underdevelopm e n t of the proventriculus a n d ventriculus. Foreign body p e n e t r a t i o n of the ventricular wall can occur in any species, but is most c o m m o n in waterfowl a n d ratites. Nutritional muscular dystrophy (degenerative myopathy) can be seen as white streaks in the ventricular muscle in some species as a manifestation of vitamin E / s e l e n i u m deficiency. Endoventricular mycosis (fungal invasion of the koilin lining of the ventriculus) can be seen histologically and is a c o m m o n finding in debilitated passerines, despite the usually unremarkable gross appearance. O p e n the outflow tract f r o m the ventriculus and p r o c e e d into the d u o d e n a l loop. The largest limb of the pancreas lies in the duodenal loop mesentery while the small splenic pancreatic lobe is located adjacent to the spleen. Quaker parrots (Myiopsitta monachus) are p r o n e to the d e v e l o p m e n t of acute pancreatic necrosis of unknown etiology. Q u a k e r parrots that survive the initial insult may develop severe pancreatic atrophy and fibrosis. Inclusion body pancreatitis can be seen with herpesvirus and adenovirus infections. Lymphoplasmacytic pancreatitis in Neophema parrots is associated with paramyxovirus infection. Pancreatic necrosis is also a comm o n lesion in West Nile virus infection. Vacuolar changes and necrosis of acinar cells may be seen in zinc toxicosis. T h e pancreas concentrates zinc in the acinar cells and should be collected for toxicologic analysis, along with liver and kidney, to diagnose this toxicity. Collect a sample of pancreas for virology. Also, submit a transverse section t h r o u g h the duodenal loop with pancreas attached in formalin, as this helps to identify the d u o d e n u m . Continue o p e n i n g the intestine through the j e j u n u m and ileum to the ceca (if present in the species) and colon. In neonates, the yolk sac and stalk should be evaluated for the degree of absorption. In psittacine and passerine chicks, the yolk sac is usually quite tiny by three days after
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hatching. Collect a sterile sample of the yolk material for culture a n d place the rest of the yolk sac (wall and contents) into formalin. Yolk sacculitis and yolk sac retention are c o m m o n p r o b l e m s in neonatal ratites. Collect sections o f intestine for histopathology. O p e n e d intestinal sections are usually best, as this gives the m u c o s a a chance to fix rapidly. Do not disturb the m u c o s a by scraping as artifacts can confuse or obliterate the histologic diagnosis. Wet mounts of intestinal contents (usually two different sites) are helpful in diagnosing parasitic and bacterial problems. Wet mounts should be e x a m i n e d for parasite ova and oocysts, as well as flagellates, yeast and motile bacteria. Sections of bowel can be tied off with string or suture and submitted for culture. In some cases, both aerobic and anaerobic culture may be warranted. Clostridial enteritis is becoming m o r e c o m m o n l y recognized in psittacines, especially nectar-eaters, such as lories and loftkeets. Clostridial organisms in large n u m b e r s can cause acute, n e c r o - h e m o r r h a g i c enteritis. Finding large Gram-positive bacilli, with or without spore formation, as the primary organism on a Gram-stained s m e a r of intestinal contents gives a presumptive diagnosis, which should be followed by anaerobic culture. A wide variety of o t h e r bacteria can cause enteritis and septicemia. Gram-negative organisms, especially of the Enterobacteriaceae, are c o m m o n in psittacines and passerines. In addition, Campylobacter spp and gersinia pseudotuberculosis are m o r e c o m m o n in canaries and exotic finches; Campylobacter organisms usually require special m e d i a and microaerophilic incubation conditions, so it is wise to alert the bacteriology laborato U when this organism is suspected. Multifocal granulomas or thickened areas of bowel can be indicative of mycobacteriosis. These sites should be collected for histopathology, and special tissue acid-fast stains can be applied to paraffin sections to d e m o n s t r a t e the organisms. Alternatively, impressions or scrapings f r o m these sites can be stained with a rapid acid-fast stain for a quick, presumptive diagnosis. Intestinal neoplasia is fortunately u n c o m m o n in birds, but needs to be included in the differential diagnosis of thickened or proliferative bowel lesions. Flagellate p r o t o z o a and coccidial organisms may also p r o d u c e enteritis. Flagellates (including Giardia spp and Cochlosoma spp) are diag-
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nosed by fresh wet m o u n t smears of intestinal contents. N e m a t o d e and cestode parasites are u n c o m m o n in domestically-raised psittacines and passerines, but geographic pockets of these parasites may exist and should always be considered. These parasites are still c o m m o n in ground-feeding and wild birds. Many species of birds do not possess ceca. Psittacines do not, passerines and Columbiformes have tiny vestigial ceca c o m p o s e d of lymp h o i d tissue, while Galliformes, Anseriformes and ratites possess large bilateral ceca. These should be o p e n e d to look for cecal worms and their contents should be included for culture. Colon contents should start to look like fecal material as one moves aborad. O p e n the cloaca to look for papillomatous lesions, cloacoliths, t r a u m a and inflammatory lesions. In summary, intestinal samples should include: wet mounts f r o m at least two different sites; smears for G r a m stain and possibly acid-fast stain; contents for aerobic, and possibly anaerobic or Campylobacter culture; and tissue and ingesta for virology (electronmicroscopy, virus isolation, or DNA probes).
Neurologic T h e brain and spinal cord can be very important in the diagnosis of some diseases, especially PDD. The dorsal calvarium should be carefully r e m o v e d with rongeurs. Visualize the brain in situ for any obvious abnormalities, such as abscesses, which should be cultured. Remove the brain by inverting the skull and transecting the ventral and cranial attachments. Collect a portion of the forebrain for virology and toxicology and fix the rest of the brain in formalin. In neonates, the brain is so soft that making a cut t h r o u g h the dorsal skull and placing the entire calvarium containing the brain in situ into formalin is r e c o m m e n d e d . After fixation, the brain will h a r d e n somewhat a n d it can m o r e easily be r e m o v e d without d a m a g i n g it. A similar procedure can be followed for the cervical spinal cord. Cut the vertebral column with cord in situ into 1 to 2 cm pieces and fix in formalin. This process will allow easier removal using rongeurs with minimal d a m a g e to the less fragile, fixed spinal cord. In birds with head tilt or neurologic disease, especially Neophema parrots and exotic finches, fix a large portion of the petrous t e m p o r a l b o n e
Practical Avian Necropsy
containing the middle ear. This b o n e can later be decalcified by tile veterinary pathologist and sectioned to examine the middle ear for inflammation a n d viral inclusions associated with paramyxovirus infections. Congestion of the vascular sinuses in the bones of the skull is a comm o n finding, but it is only significant if there is also c o r r e s p o n d i n g subdural h e m o r r h a g e or bleeding of brain parenchyma.
Musculoskeletal Bone m a r r o w can be collected by aspiration of the f e m u r and smears m a d e and stained for cytologic evaluation. Collect a segment of f e m u r using rongeurs and place in formalin. O n c e fixed, the previously fragile b o n e marrow can be dissected out and e x a m i n e d histologically. Leukemic or aplastic processes can be diagnosed f r o m b o n e m a r r o w samples and occasionally circovirus inclusions may also be seen histologically. Samples of skeletal muscle should be collected for histopathology. Muscular lesions may include trauma, h e m o r r h a g e , degeneration, mineralization and injection or vaccine site reactions. Myositis, degenerative myopathy, a n d Sarcocystis infection can be diagnosed histologically. O p e n the joints of the pelvic and thoracic limbs a n d look for exudate (often caseous), gouty tophi, and lesions of degenerative j o i n t disease. Any b o n e or j o i n t lesions d e m o n s t r a t e d radiographically should be o p e n e d and sampled for culture and histopathology. The flexibility of bones (tibiotarsus, ribs, etc.) can be used to assess adequate mineralization. The rachitic 'rosary' at the costochondral or costovertebraljunctions and d e f o r m a t i o n of the keel or o t h e r long bones are obvious lesions of metabolic b o n e disease. T h e bones should b r e a k with an audible snap if mineralization is normal. Sections of bone, especially areas of the metaphyses a n d epiphyses, can be e x a m i n e d histologically for metabolic b o n e disease.
Final Comments This completes the gross necropsy and the r e m a i n i n g parts of the carcass can be placed in a sealable plastic bag a n d frozen until diagnostic testing has b e e n completed. Examine wet m o u n t s as quickly as possible. Stain any exudates
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Table 1. In Birds, Collect the Following Tissues for Histopathology if No Gross Lesions Are Present Heart Liver Kidney Lung Brain Bursa (in young birds)
a n d / o r impression smears collected. Perform G r a m stains as indicated. A collection of liver, spleen, a n d airsac can be sent for Chlamydophila diagnostics (Gimenez, PCR, fluorescent antibody assay, culture, etc). Send tissues, exudates or swabs for bacterial, mycoplasmal or fungal culture as indicated. With the exception of samples for Campylobacte~; which does not survive freezing well, these samples can often be frozen if not sent for culture immediately. A pool of parenchymal tissues (liver, spleen, airsac, lung, kidney, brain) and a separate pool of intestinal contents should be refrigerated or frozen for possible virus isolation or DNA p r o b e testing. Fluorescent antibody techniques on frozen sections of tissue may be available for certain viruses. Select a g r o u p of formalin-fixed tissues with lesions or a g r o u p of tissues that c o m m o n l y contain histologic lesions that could lead to diagnosis and submit t h e m for histopathology. This c o m m o n l y includes tissues such as liver, spleen, airsac, kidney, lung, trachea, heart, bursa, brain, d u o d e n u m / p a n c r e a s , and proventriculus/ventriculus. Table 1 lists tissues that should be collected if no gross lesions are seen. Save the r e m a i n i n g formalin-fixed tissues, just in case the diagnosis is not m a d e with the first set. The veterinary pathologist may r e c o m m e n d special diagnostics, such as stains for acid fast organisms, fungi, bacteria, iron, copper, etc., d e p e n d i n g on what is seen on the routine hematoxylin and eosin-stained sections. In special situations, tissues may be e m b e d d e d in plastic so that electron microscopy can be p e r f o r m e d . Direct electronmicroscopy can also be p e r f o r m e d on intestinal contents or tissue homogenates. In situ DNA hybridization techniques on paraffine m b e d d e d tissues are available for certain viruses, such as Pacheco's herpesvirus, adenovirus, avian polyomavirus, psittacine circovirus, and paramyxovirus. I m m u n o h i s t o c h e m i c a l stains
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c a n d e t e c t c e r t a i n a n t i g e n s f r o m bacteria, f u n g i , viruses, a n d parasites a n d these t e c h n i q u e s c a n also b e utilized to i d e n t i f y s o m e cell m a r k e r s i n the diagnosis o f t u m o r s . T o x i c o l o g i c testing r e q u i r e s s o m e i d e a o f what t o x i n is b e i n g c o n s i d e r e d . This i n f o r m a t i o n o f t e n c o m e s f r o m the history a n d h i s t o p a t h o logic findings. C o n t a c t i n g the toxicology laboratory is essential for s u b m i s s i o n of the m o s t app r o p r i a t e tissues a n d a m o u n t s . T h e m o s t comm o n toxins tested are heavy metals, such as lead a n d zinc. Usually liver a n d k i d n e y are r e q u i r e d for this analysis, a l t h o u g h zinc a c c u m u l a t e s i n the p a n c r e a s preferentially. P o i s o n o u s p l a n t s c a n b e f o u n d i n the digestive tract a n d s u b m i t ted to a b o t a n i s t or university b o t a n y d e p a r t m e n t for i d e n t i f i c a t i o n . P o l y t e t r a f l u o r e t h y l e n e (Teflon) a n d o t h e r toxic i n h a l a t i o n p r o d u c t s are rarely d e t e c t a b l e i n tissues, a n d the diagnosis is usually m a d e with history o f e x p o s u r e , the presence of p u l m o n a r y e d e m a and hemorrhage, a n d e x c l u s i o n o f o t h e r causes o f death. Mycotoxins m a y be i m p l i c a t e d i n the case o f m u l t i p l e birds suffering liver d a m a g e . Aflatoxins c a n be detected i n foodstuffs, b u t usually by the time c h r o n i c liver d a m a g e is evident, the o f f e n d i n g f o o d s t u f f is o f t e n n o l o n g e r available. I n the case of a c u t e toxicosis, freeze samples of tile feed a l o n g with liver a n d kidney, p e n d i n g f u r t h e r investigation. I n c o n c l u s i o n , gross n e c r o p s y a n d p o s t m o r t e m d i a g n o s t i c testing is a very i m p o r t a n t p a r t o f avian m e d i c i n e , r e q u i r i n g a systematic a p p r o a c h to the e x a m i n a t i o n o f o r g a n s a n d the c o l l e c t i o n o f samples. D e v e l o p i n g a r e l a t i o n s h i p with a vete r i n a r y p a t h o l o g i s t is necessary to s u b m i t the a p p r o p r i a t e s a m p l e s a n d to o p t i m i z e the c h a n c e s for a r r i v i n g at a diagnosis.
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Recommended Reading 1. Altman RB, Forbes NA: Self-AssessmentColor Review of Avian Medicine. Ames, LA, Iowa State University Press, 1998 2. Campbell TW: Avian Hematology and Cytology (ed 2). Ames, IA, Iowa State University Press, 1995 3. Dorrestein GM: Diagnostic necropsy and pathology, in Altman RF, Clubb SL, Dorrestein GM, Quesenberry K (eds): Avian Medicine and Surgery. Philadelphia, PA, Saunders, 1997, pp 158-169 4. Fudge AM: Laboratory Medicine: Avian and Exotic Pets. Philadelphia, PA, Saunders, 2000 5. Graham DL: Necropsy techniques for pet birds, in 1986 Scientific Proceedings, 54th Annual Meeting of the American Animal Hospital Association. Denver, CO, American Animal Hospital Association, 1987, pp 455 6. Latimer KS, Rakich PM: Necropsy examination, in Ritchie BW, Harrison GJ, Harrison LR (eds): Avian Medicine: Principles and Application. Lake Worth, FL, Wingers Publishing, 1994, pp 355-379 7. Lowenstine LJ: The postmortem examination, in Proceedings of the Parrot Biology Symposium, Psittacine Research Project. Davis, CA, 1992, pp 37-42 8. Randall CJ: A Colour Atlas of Diseases of the Domestic Fowl and Turkey. Ames, IA, Iowa State University Press, 1985 9. Randall CJ, Reece RL: Color Atlas of Avian Histopathology. London, Mosby-Wolfe, 1996 10. Reavill D, Schmidt RE, Garner M: How to get the most for your pathology dollar. Exotic DVM 1(6):33-40, 2000 11. Reavill D: Getting an answer: tips on how to improve diagnostic testing, in Proceedings of the Annual Conference of the Association of Avian Veterinarians. Boca Raton, FL, Association of Avian Veterinarians, 2002, pp 271-278 12. Riddell C: Avian Histopathology (ed 2). Kennett Square, PA, American Association of Avian Pathologists, 1996 13. Ritchie BW: AvianViruses: Function and Control. Lake Worth, FL, Wingers Publishing, 1995 14. Samonr J: Avian Medicine. London, Mosby-Harcourt Publishers, 2000 15. Schmidt RE: 'Sudden' death in pet birds, in Proceedings of the Annual Conference of the Association of Avian Veterinarians. Boca Raton, FL Association of Avian Veterinarians, 1995, pp 473-478
This article is a review of the necropsy techniques utilized in birds with emphasis on psittacine and passerine species. The aim of this article is to assist the practitioner in systematically performing a gross necropsy in the veterinary practice setting and collecting and submitting appropriate samples with the goal of arriving at meaningful diagnoses. Common disease entities are discussed along with the tissues and other samples required for definitive diagnosis of these disorders. 9 2003 Elsevier Inc. All rights reserved. Key words: Avian, necropsy, diagnostics, technique, post-mortem.
he necropsy examination is an i m p o r t a n t part of avian medicine. Often, clinical signs a n d clinical p a t h o l o g i c findings are n o t definitively e x p l a i n e d until necropsy. N e c r o p s y exa m i n a t i o n iS m u c h m o r e t h a n satisfying the curiosity of the owner, b r e e d e r or attending veterinarian. It provides i m p o r t a n t information that can be used in the diagnosis and t r e a t m e n t of future cases. Necropsy findings are an integral part of the flock database f r o m which husbandry, m a n a g e m e n t , treatment, vaccination and quarantine r e c o m m e n d a t i o n s can be made. P o s t m o r t e m information can be invaluable in educating owners of the seriousness of husbandry, nutritional and infectious disease conditions and thereby prevent owners f r o m making the same mistakes in subsequent pets. For the grieving owner, necropsy findings can relieve some or all of the guilt associated with the death of a beloved pet, assure t h e m that they did all they reasonably could, and provide a step in the process toward closure. In situations where the owner may be dissatisfied with t r e a t m e n t or outcomes, it is wise to have a veterinary pathologist p e r f o r m the necropsy. The veterinary patholo-
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From Amazon Veterinary Diagwostics, Salem, OR. Address reprint requests to Madeline A. Rae, DVM, MS, DABVP (Avian Practice), Amazon Veterinary Diagnostics; P.O. Box 7790, Salem, OR 97303-1075. 9 2003 Elsevier Inc. All rights reserved. 1055-937X/03/1202-0002530. 00/0 doi: 10.1053/saep. 2003.127882
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gist should have training, experience and interest in pet and wild birds. T h e necropsy should be p e r f o r m e d as soon after death as is possible. To prevent dry feathers f r o m insulating the body and delaying cooling, wet the feathers with a detergent and water solution. T h e body should be refrigerated, not frozen. Freezing can create artifacts in the tissues that may seriously obscure histologic lesions. P o s t m o r t e m autolysis can also obscure histologic lesions, so if necropsy c a n n o t be perf o r m e d within 3 days, the body should be frozen, realizing that histopathology is likely to be compromised. W h e n shipping a cooled body, be sure the ice-packs (or other frozen coolants) do not directly touch the body, as this may freeze the body tissues, especially in very small birds. Wrap the ice-packs in bubble-wrap or newspaper to prevent freeze damage. Obtain a detailed history, including age of the bird, diet, environment, recent additions to the flock or household, as well as signs of illness. Include any t r e a t m e n t used and if the bird was euthanized, indicate how this was performed. Since intracoelomic injections of barbiturate euthanasia solutions can create extensive lesions, the best euthanasia m e t h o d is to use an overdose of an inhalant anesthetic gas, as this leaves the least a m o u n t of artifactual changes in the body. Even intravenous injections of barbiturates can result in widespread lysis of erythrocytes with pooling of solution in the heart, thereby causing iatrogenic lesions. Instrmnents for the avian necropsy are few. They include a scalpel and handle, thumb-forceps, scissors, a n d rongeur-type i n s t r u m e n t for cutting b o n e and removing the brain. A set of ophthlamic instruments and a h e a d loupe are invaluable in the necropsy of small passerines, neonates or dead-in-shell embryos. Autoclave two sets of the instruments (the rongeurs are only n e e d e d in one set); use one set for o p e n i n g the bird a n d the o t h e r to collect internal organ samples aseptically for culture a n d virus isolation. Microscope slides, coverslips, sealable plastic bags, and formalin bottles or jars should also
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be available. Be sure that formalin fumes do not contact tissues that are to be cultured for bacteria or viruses as this can compromise the culture accuracy. Make sure that formalin fumes do not come in contact with blood or tissue cytological smears as this can severely distort staining and interpretation. Perform the necropsy in a well-lighted, wellventilated area (preferably u n d e r a fume hood) and wear gloves, a mask and if possible a disposable apron. Aerosols from feathers, feces and exudates can be infectious. This is particularly important with cases of chlamydophilosis and mycobacteriosis, which can be zoonotic. However, it is also important to contain the feather d a n d e r and feces in cases of avian polyomavirus and psittacine circovirus infections, so as not to contaminate the premises, your clothing or other adjacent birds. Disinfectant solutions should be readily available for clean-up after the necropsy, but these solutions, nor their fumes, should come in contact with tissues being collected, as they may lyse cells and destroy microorganisms n e e d e d for culture. The particular routine used for gross necropsy of birds can vary, but what remains the same is that all organs are examined. The use of a checklist will ensure that all organs and systems are examined; d o c u m e n t all findings and make this checklist a part of the medical record. It is important to collect samples of everything (all organs, the grossly normal and abnormal). After the necropsy is completed, the decision of which samples to send and what tests to request can be made, but at the very least, the diagnosis does not get cremated with the carcass. Before the necropsy, label sealable bags and formalin jars with the owner's name and the tissues enclosed in an effort to save time and prevent interruptions in the flow of the necropsy.
External Examination The necropsy begins with an external examination. Record the band n u m b e r and scan for microchips; these can be removed, labeled, and saved as p r o o f of identification. Palpate for obvious fractures; in some instances radiographs may be warranted. Examine the skin and feathers. Often feather abnormalities may not be visible while the feather remains in the follicle. For example, the concentric pinching of the feather
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shaft, seen in psittacine circovirus infection, may not be visualized until the feather is plucked from the follicle. Look for stress bars in the wing and tail primaries. Collect multiple blood feathers, both plucked and in the follicle, along with any skin lesions, and place them in formalin. Check for any signs of trauma or bruising. In neonates, closely examine the umbilicus for cleanliness and the adequacy of healing. Examine the unfeathered portions of the legs and the feet for poxvirus lesions, bumblefoot, herpesvirus pododermatitis, and self-mutilation. Examine the uropygial gland, f o u n d at the base of the tail in some species and collect it for histopathologic evaluation, as this can be a site of chronic inflammation and neoplasia. Evaluate the beak, both the external and the intraoral surfaces. O p e n the mouth. Look at and u n d e r the tongue for abnormalities. Look in the choanal slit for mucus and exudate and for blunting of the choanal papillae. Salivary gland enlargement can occur at the base of the tongue and can be due to hypovitaminosis A, bacterial abscesses, or rarely mycobacterial infections. The nares and ear canals should be clear and free of debris or exudate. Examine the conjunctivae and the nictitating membranes. In Columbiformes, these tissues can be collected for Chlamydophila diagnostics, as they may contain elementary bodies. The infra-orbital sinuses should be o p e n e d as aseptically as possible and swabs or aspirates collected for cytology and culture of routine bacteria, Mycoplasma and fungi. Bacterial sinusitis is quite c o m m o n in psittacines, but also occurs in passerine species, and caseous exudate is often seen. In cockatiels (Nymphicus hoUandicus) with 'lockjaw,' sinusitis and temporomandibulitis is c o m m o n , as well as myositis of the mandibular muscles. The mandible and its attached muscles can be placed in formalin for histopathology. In these cases, bacteria such as Bordetella avium, E.nterococcus, E. coli and Enterobacter may be isolated. It is important to indicate to the bacteriology laboratory that Bordetella avium is suspected because this organism is somewhat fastidious and colonies may take longer to appear. Bordetella avium may also cause tracheitis, bronchitis, and p n e u m o n i a in cockatiels and rarely in other psittacines. Several Mycoplasma species have been implicated in conjunctivitis and sinusitis in psittacines and passe-
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rines, but these require special media for isolation and are recovered uncommonly.
Coelomic Cavity The coelomic cavity examination is begun by placing the body in dorsal recumbency, incising the skin of the a b d o m e n and peeling it back caudally over the a b d o m e n and cranially over the pectoral muscle mass. Assess the condition of the pectoral muscle, as this is a good measure of weight loss. Notice whether subcutaneous fat is present or absent and whether there is any bruising or edema. Grasp the sternum with thumb forceps and slightly elevate, maintaining tension on the abdominal skin. Using a scalpel blade, make a transverse incision just caudal to the edge of the sternmn, being careful not to lacerate the liver. Remove the keel and pectoral muscles in one piece by cutting through the ribs and shoulder girdle.
Liver and Spleen Assess the size, color, and consistency of the liver. Hepatic margins should be sharp and not extend beyond the caudal edge of the keel. Note whether a gall bladder is present or absent, as not all species have one. Assess the condition of the airsacs and peritoneal reflections that are visible. If coelomic fluid is present, collect it with a sterile syringe for analysis. Collect samples of airsacs aseptically next, since they are delicate structures that readily disappear with further manipulation of the organs. Aseptically collect liver samples: one sample each for bacteriology, virus isolation or DNA probe testing, Chlamydophila testing, and histopathology. Use any remaining tissue for toxicology a n d / o r impression smears. Grasp the ventriculus, elevate and incise through the attached m e m b r a n e / a i r s a c and then rotate the ventriculus counterclockwise to find the spleen. The spleen is nestled in the cmwe between the proventriculus and the ventriculus. Evaluate the size and shape of the spleen. Determining whether it is of normal size for the bird being necropsied requires some practice, so measuring the diameter can be helpful. The spleen is r o u n d in some species, such as Psittaciformes and Galliformes, and elongated or sock-like in Passeriformes and Columbi-
formes. Note color and any pale foci in the spleen. Collect the entire spleen, dividing it into three samples: one each for virology, Chlamydophila diagnostics, and histopathology. The spleen is the single most important sample for the histopathologic diagnosis o f avian polyomavirus infection, since this is where viral inclusions are most abundant.
Genitourinary Reflect the ventriculus and the intestinal tract to the right side of the bird to view the adrenals, gonads and kidneys leaving the u n o p e n e d gastrointestinal tract for last to avoid contamination of the other abdominal organs. The adrenals are often obscured by active gonadal tissue, so it is easier to collect the cranial division of the kidney with the adrenal and gonad(s) attached for histopathology. Adrenalitis is sometimes noted in unexplained death and may be the only abnormality in some psittacines with proventricular dilation disease (PDD). Sex the bird visually. In most species, only the left ovary and oviduct develop in females, but both testes develop in male birds. The gonads may be pigmented (brown or black) in some species, most notably cockatoos. Note the degree of development of the ovary and oviduct. Is there follicular development? Is so, record the general size of the follicles. Is the oviduct hypertrophied? O p e n the oviduct to look for exudate and tumors, and collect samples for bacteriology and histopathology as needed. Testicular tumors are c o m m o n in budgerigars (Melopsittacus undulatus) and ducks, but seasonal testicular enlargem e n t also occurs and in some species, such as in many passerine birds, can be mistaken for neoplasia; histopathology can usually distinguish between these two changes. The kidneys are nestled in the renal fossae of the synsacrum, with the lumbosacral plexus lying deep to the caudal division of the kidney. The ureters run down the ventral surface of the kidneys bilaterally. In addition to the kidney/adren a l / g o n a d tissue collected above for histopathology, aseptically collect additional renal samples for virology, toxicology, and bacteriology (if exudate is present). In small birds ( u n d e r 30 g), one can make an en bloc excision of the kidneys still in situ within the synsacrum and place this in formalin. After fixation, renal dissection is easier
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metabolic b o n e disease needs to be considered. Parathyroid h y p e r t r o p h y is usually the only gross pathologic lesion f o u n d in the hypocalcemia syndrome of African gray parrots (Psittacus er-
ithacus)
Cardiac
Figure 1. Anatomic location of normal avian thyroids with the keel removed. Thyroids (a) are located on either side of the trachea. The normal parathyroids are barely visible as they are usually closely associated with the caudal poles of the thyroids. Trachea (b). Carotid arteries (c). Heart (d). a n d / o r the synsacrum can be decalcified a n d cross-sections of kidney together with b o n e can be cut. After removal of the kidneys, evaluate the lumbosacral plexus, especially in cases of pelvic limb weakness or malfunction; samples of these nerves can be collected in formalin for histopathologic evaluation.
Thoracic Inlet Move to the thoracic inlet region. Identify the thyroids a n d parathyroids, located cranial to the heart and adjacent to the carotid arteries bilaterally, a n d collect t h e m for histopathology. (Fig 1) Goiterous changes were once quite c o m m o n in budgerigars, but are less so with the advent of c o m m e r c i a l diets. Hyperplastic goiter has b e e n seen in juvenile macaws recently. Lymphocytic thyroiditis may also be seen histologically, especially in A m a z o n parrots. N o r m a l parathyroids are barely visible. W h e n they are p r o m i n e n t ,
Examine the heart, p e r i c a r d i u m and great vessels. Visceral gout can cause the deposition of white, m u c o i d urate material on the pericardial sac. View a s m e a r of this material u n d e r polarized light to confirm the presence of uric acid crystals. Formalin fixation may dissolve uric acid crystals a n d they may not be visible on histopathology (crystals do not dissolve if fixed in ethanol, but this is usually not a practical fixative for other reasons). Suppurative pericarditis can be caused by a variety of bacteria, such as Pasteurella and Chlamydophila. Cytologic examination of the pericardial exudate may reveal the causative organisms. H y d r o p e r i c a r d i u m is a c o m m o n finding in avian polyomavirus infection in juvenile psittacines. Before removing the heart f r o m the thoracic cavity, heart b l o o d can be collected using a sterile syringe a n d needle for bacteriology. Smears of heart b l o o d can be stained with Wright's stain and e x a m i n e d for h e m o p r o t o z o a and microfilaria, or G r a m stained to look for bacteria. After removing the heart and great vessels, o p e n the heart in the direction of blood flow, using water to rinse away b l o o d and clots. L o o k for thrombi, valvular endocarditis lesions, a n d pale areas in the myocardium. Congenital cardiac anomalies are rare. O p e n the great vessels to look for atherosclerosis, which may involve the aorta, p u l m o n a r y artery or carotids. Atherosclerosis is characterized grossly by yellowish, raised, intimal plaques, but may occasionally be so severe that the carotids are completely obstructed. Mineralization of the great vessels may also occur in association with atherosclerosis or related to renal disease and hypervitaminosis D. Atherosclerosis is most c o m m o n l y seen in African gray parrots, where it can be mild to moderate, but also in obese, older Amazon parrots (Amazona spp.) a n d captive raptots, where it can be so severe that it results in acute death. Atherosclerotic lesions may also be f o u n d in the coronary arteries, but usually these
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lesions are discovered on histopathologic examination. It is best to place most of the heart in formalin, so that multiple sections can be taken for histopathologic evaluation. Pale loci or streaks can indicate degenerative m y o p a t h y related to vitamin E / s e l e n i u m deficiency or myocarditis associated with septicemia or viral diseases, such as West Nile virus or PDD. Petechial and ecchymotic epicardial h e m o r r h a g e s are c o m m o n l y seen in cases of acute death f r o m avian polyomavirus.
Respiratory Examine the lungs in situ before removing them. Avian lungs are fixed in place within the avian thoracic cavity, and are not freely moveable. Removal requires gentle teasing of the lung tissue away f r o m the ribs. The avian lung is one tissue in which gross lesions may a p p e a r quite significant, but on histopathologic evaluation turn out to be just passive congestion. Conversely, grossly n o r m a l lungs may contain significant histologic lesions. So, it is wise to always include lung for histopathology, even if it appears grossly normal. Collect a portion of the lung for bacteriology and virology and place the rest in formalin for histopathology. Because lesions can be focal or multifocal, it is best to include a large portion of at least one lung. In canaries and mynahs, a Wright's-stained impression s m e a r of lung (along with impressions from liver and spleen) is very i m p o r t a n t in detecting the monocytic f o r m of atoxoplasmosis, as Atoxoplasma organisms are not visible on histopathology. Sarcocystis organisms can also be seen in impression smears of the lung.
Oral Cavity and Gastrointestinal Tract Cutting t h r o u g h the mandible at one of the lateral commissures allows access to the caudal p h a r y n x and visualization of the glottis. T h e larynx and trachea can then be o p e n e d down to the tracheal bifurcation, looking for hemorrhage, exudate, foreign bodies, granulomas, and parasites, such as respiratory mites or Syngamus nematodes. Tissue samples should be collected for histopathology and virus isolation, as well as
bacteriology and fungal culture if warranted. Fungal tracheitis, especially at the syrinx, can be diagnosed by cytologic examination of exudate or g r a n u l o m a t o u s material, fungal culture, a n d / o r by histopathology. Rarely, syringeal granulomas can be caused by mycobacterial organisms. In canaries, Enterococcus faecalis can cause chronic tracheobronchitis; culture of the tracheal l u m e n is necessary for diagnosis. Viral tracheitis is rare in psittacine birds, but a herpesviral tracheitis, bronchitis and airsacculitis has b e e n r e p o r t e d in Neophema parrots. Canarypox can p r o d u c e a severe tracheobronchitis with intracytoplasmic inclusions. Going back to the pharynx, the cut can extend downward the length of the esophagus and into the crop, looking for lacerations a n d punctures, peri-esophageal abscesses, and o t h e r abnormalities. In game birds, the esophagus and crop may exhibit m o d e r a t e to m a r k e d thickening and m u c u s p r o d u c t i o n due to capillariasis. A wet m o u n t scraping f r o m the crop can reveal the typical bipolar capillarid ova. In juvenile psittacines, thickening and 'Turkish towel' app e a r a n c e of the crop mucosa is often due to candidiasis and either a wet m o u n t smear, cytology or G r a m stain of a crop mucosal scraping can be diagnostic. T r i c h o m o n a d s can be f o u n d in wet m o u n t s f r o m the oral cavity a n d / o r crop of Columbiformes and raptors, but may also occasionally b e f o u n d in the crops of budgerigars and passerines. T h e crop contents can be collected in a plastic bag and frozen, if there is any suggestion o f a toxin ingestion. A large section of crop, to include a big vessel and adjacent nerve, should be collected for histopathology, since PDD lesions are often confined to the nerves. At this point, the esophagus distal to the crop can be transected. Caudal traction of the distal esophagus and sharp dissection of the mesenteric attachments can be used to remove the entire gastro-intestinal tract. Continue the dissection to m a k e a circular incision a r o u n d the vent, leaving a margin of intact vent skin and the bursa of Fabricius attached to the tract. The bursa is present in young birds, usually less than 6 to 12 m o n t h s o f age, and is located dorsal to the cloaca. (Fig 2) The bursa should always be collected w h e n it is present and divided in hale Submit one half in formalin for histopathology a n d save the other half for virology a n d / o r DNA
Practical Avian Necropsy
Figure 2. Anatomic location of the normal avian bursa in a 4-month-old Amazon parrot (Amazona sp). The bursa (a) is located dorsal to the cloaca. The colon (b) is retracted caudally to expose the bursa. The caudal division of the kidney is noted (c).
p r o b e testing. T h e bursa is i m p o r t a n t in diagnosing psittacine circovirus, especially in young African gray parrots that die acutely without feather lesions, since the bursa may be the only site where viral inclusions are found. Lesions in the bursa are often nonspecific as to etiology, but can indicate the acuteness or chronicity of stress. O p e n the distal esophagus with scissors, continuing on into the proventriculus and ventriculus. Evaluate the stomach contents for a m o u n t and foreign material. Collect and freeze the contents for possible toxicologic analysis. Rinse the mucosa with water and m a k e wet m o u n t and dried smears of m u c u s a n d / o r mucosal scrapings. Do not separate the proventriculus and ventriculus. T h e isthmus (the junction between the proventriculus and ventriculus) is a c o m m o n site for avian gastric yeast (formerly known as megabacteria; suggested new name, Virgamyces avigast'r and gastric carcinoma. Collect a large specimen of proventriculus, isthmus, and ventriculus (all in one piece, if possible), con-
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taining at least one large serosal nerve and blood vessel, for histopathology. A large specimen allows multiple sections to be e x a m i n e d by the veterinary pathologist in the search for nerves a n d plexi. Dilation of the proventriculus a n d / o r ventriculus is a hallmark gross lesion of PDD, but in juvenile psittacines being hand-fed, these organs may also be dilated as a n o r m a l finding. Histopathology is required to differentiate between PDD and n o r m a l juvenile underdevelopm e n t of the proventriculus a n d ventriculus. Foreign body p e n e t r a t i o n of the ventricular wall can occur in any species, but is most c o m m o n in waterfowl a n d ratites. Nutritional muscular dystrophy (degenerative myopathy) can be seen as white streaks in the ventricular muscle in some species as a manifestation of vitamin E / s e l e n i u m deficiency. Endoventricular mycosis (fungal invasion of the koilin lining of the ventriculus) can be seen histologically and is a c o m m o n finding in debilitated passerines, despite the usually unremarkable gross appearance. O p e n the outflow tract f r o m the ventriculus and p r o c e e d into the d u o d e n a l loop. The largest limb of the pancreas lies in the duodenal loop mesentery while the small splenic pancreatic lobe is located adjacent to the spleen. Quaker parrots (Myiopsitta monachus) are p r o n e to the d e v e l o p m e n t of acute pancreatic necrosis of unknown etiology. Q u a k e r parrots that survive the initial insult may develop severe pancreatic atrophy and fibrosis. Inclusion body pancreatitis can be seen with herpesvirus and adenovirus infections. Lymphoplasmacytic pancreatitis in Neophema parrots is associated with paramyxovirus infection. Pancreatic necrosis is also a comm o n lesion in West Nile virus infection. Vacuolar changes and necrosis of acinar cells may be seen in zinc toxicosis. T h e pancreas concentrates zinc in the acinar cells and should be collected for toxicologic analysis, along with liver and kidney, to diagnose this toxicity. Collect a sample of pancreas for virology. Also, submit a transverse section t h r o u g h the duodenal loop with pancreas attached in formalin, as this helps to identify the d u o d e n u m . Continue o p e n i n g the intestine through the j e j u n u m and ileum to the ceca (if present in the species) and colon. In neonates, the yolk sac and stalk should be evaluated for the degree of absorption. In psittacine and passerine chicks, the yolk sac is usually quite tiny by three days after
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hatching. Collect a sterile sample of the yolk material for culture a n d place the rest of the yolk sac (wall and contents) into formalin. Yolk sacculitis and yolk sac retention are c o m m o n p r o b l e m s in neonatal ratites. Collect sections o f intestine for histopathology. O p e n e d intestinal sections are usually best, as this gives the m u c o s a a chance to fix rapidly. Do not disturb the m u c o s a by scraping as artifacts can confuse or obliterate the histologic diagnosis. Wet mounts of intestinal contents (usually two different sites) are helpful in diagnosing parasitic and bacterial problems. Wet mounts should be e x a m i n e d for parasite ova and oocysts, as well as flagellates, yeast and motile bacteria. Sections of bowel can be tied off with string or suture and submitted for culture. In some cases, both aerobic and anaerobic culture may be warranted. Clostridial enteritis is becoming m o r e c o m m o n l y recognized in psittacines, especially nectar-eaters, such as lories and loftkeets. Clostridial organisms in large n u m b e r s can cause acute, n e c r o - h e m o r r h a g i c enteritis. Finding large Gram-positive bacilli, with or without spore formation, as the primary organism on a Gram-stained s m e a r of intestinal contents gives a presumptive diagnosis, which should be followed by anaerobic culture. A wide variety of o t h e r bacteria can cause enteritis and septicemia. Gram-negative organisms, especially of the Enterobacteriaceae, are c o m m o n in psittacines and passerines. In addition, Campylobacter spp and gersinia pseudotuberculosis are m o r e c o m m o n in canaries and exotic finches; Campylobacter organisms usually require special m e d i a and microaerophilic incubation conditions, so it is wise to alert the bacteriology laborato U when this organism is suspected. Multifocal granulomas or thickened areas of bowel can be indicative of mycobacteriosis. These sites should be collected for histopathology, and special tissue acid-fast stains can be applied to paraffin sections to d e m o n s t r a t e the organisms. Alternatively, impressions or scrapings f r o m these sites can be stained with a rapid acid-fast stain for a quick, presumptive diagnosis. Intestinal neoplasia is fortunately u n c o m m o n in birds, but needs to be included in the differential diagnosis of thickened or proliferative bowel lesions. Flagellate p r o t o z o a and coccidial organisms may also p r o d u c e enteritis. Flagellates (including Giardia spp and Cochlosoma spp) are diag-
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nosed by fresh wet m o u n t smears of intestinal contents. N e m a t o d e and cestode parasites are u n c o m m o n in domestically-raised psittacines and passerines, but geographic pockets of these parasites may exist and should always be considered. These parasites are still c o m m o n in ground-feeding and wild birds. Many species of birds do not possess ceca. Psittacines do not, passerines and Columbiformes have tiny vestigial ceca c o m p o s e d of lymp h o i d tissue, while Galliformes, Anseriformes and ratites possess large bilateral ceca. These should be o p e n e d to look for cecal worms and their contents should be included for culture. Colon contents should start to look like fecal material as one moves aborad. O p e n the cloaca to look for papillomatous lesions, cloacoliths, t r a u m a and inflammatory lesions. In summary, intestinal samples should include: wet mounts f r o m at least two different sites; smears for G r a m stain and possibly acid-fast stain; contents for aerobic, and possibly anaerobic or Campylobacter culture; and tissue and ingesta for virology (electronmicroscopy, virus isolation, or DNA probes).
Neurologic T h e brain and spinal cord can be very important in the diagnosis of some diseases, especially PDD. The dorsal calvarium should be carefully r e m o v e d with rongeurs. Visualize the brain in situ for any obvious abnormalities, such as abscesses, which should be cultured. Remove the brain by inverting the skull and transecting the ventral and cranial attachments. Collect a portion of the forebrain for virology and toxicology and fix the rest of the brain in formalin. In neonates, the brain is so soft that making a cut t h r o u g h the dorsal skull and placing the entire calvarium containing the brain in situ into formalin is r e c o m m e n d e d . After fixation, the brain will h a r d e n somewhat a n d it can m o r e easily be r e m o v e d without d a m a g i n g it. A similar procedure can be followed for the cervical spinal cord. Cut the vertebral column with cord in situ into 1 to 2 cm pieces and fix in formalin. This process will allow easier removal using rongeurs with minimal d a m a g e to the less fragile, fixed spinal cord. In birds with head tilt or neurologic disease, especially Neophema parrots and exotic finches, fix a large portion of the petrous t e m p o r a l b o n e
Practical Avian Necropsy
containing the middle ear. This b o n e can later be decalcified by tile veterinary pathologist and sectioned to examine the middle ear for inflammation a n d viral inclusions associated with paramyxovirus infections. Congestion of the vascular sinuses in the bones of the skull is a comm o n finding, but it is only significant if there is also c o r r e s p o n d i n g subdural h e m o r r h a g e or bleeding of brain parenchyma.
Musculoskeletal Bone m a r r o w can be collected by aspiration of the f e m u r and smears m a d e and stained for cytologic evaluation. Collect a segment of f e m u r using rongeurs and place in formalin. O n c e fixed, the previously fragile b o n e marrow can be dissected out and e x a m i n e d histologically. Leukemic or aplastic processes can be diagnosed f r o m b o n e m a r r o w samples and occasionally circovirus inclusions may also be seen histologically. Samples of skeletal muscle should be collected for histopathology. Muscular lesions may include trauma, h e m o r r h a g e , degeneration, mineralization and injection or vaccine site reactions. Myositis, degenerative myopathy, a n d Sarcocystis infection can be diagnosed histologically. O p e n the joints of the pelvic and thoracic limbs a n d look for exudate (often caseous), gouty tophi, and lesions of degenerative j o i n t disease. Any b o n e or j o i n t lesions d e m o n s t r a t e d radiographically should be o p e n e d and sampled for culture and histopathology. The flexibility of bones (tibiotarsus, ribs, etc.) can be used to assess adequate mineralization. The rachitic 'rosary' at the costochondral or costovertebraljunctions and d e f o r m a t i o n of the keel or o t h e r long bones are obvious lesions of metabolic b o n e disease. T h e bones should b r e a k with an audible snap if mineralization is normal. Sections of bone, especially areas of the metaphyses a n d epiphyses, can be e x a m i n e d histologically for metabolic b o n e disease.
Final Comments This completes the gross necropsy and the r e m a i n i n g parts of the carcass can be placed in a sealable plastic bag a n d frozen until diagnostic testing has b e e n completed. Examine wet m o u n t s as quickly as possible. Stain any exudates
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Table 1. In Birds, Collect the Following Tissues for Histopathology if No Gross Lesions Are Present Heart Liver Kidney Lung Brain Bursa (in young birds)
a n d / o r impression smears collected. Perform G r a m stains as indicated. A collection of liver, spleen, a n d airsac can be sent for Chlamydophila diagnostics (Gimenez, PCR, fluorescent antibody assay, culture, etc). Send tissues, exudates or swabs for bacterial, mycoplasmal or fungal culture as indicated. With the exception of samples for Campylobacte~; which does not survive freezing well, these samples can often be frozen if not sent for culture immediately. A pool of parenchymal tissues (liver, spleen, airsac, lung, kidney, brain) and a separate pool of intestinal contents should be refrigerated or frozen for possible virus isolation or DNA p r o b e testing. Fluorescent antibody techniques on frozen sections of tissue may be available for certain viruses. Select a g r o u p of formalin-fixed tissues with lesions or a g r o u p of tissues that c o m m o n l y contain histologic lesions that could lead to diagnosis and submit t h e m for histopathology. This c o m m o n l y includes tissues such as liver, spleen, airsac, kidney, lung, trachea, heart, bursa, brain, d u o d e n u m / p a n c r e a s , and proventriculus/ventriculus. Table 1 lists tissues that should be collected if no gross lesions are seen. Save the r e m a i n i n g formalin-fixed tissues, just in case the diagnosis is not m a d e with the first set. The veterinary pathologist may r e c o m m e n d special diagnostics, such as stains for acid fast organisms, fungi, bacteria, iron, copper, etc., d e p e n d i n g on what is seen on the routine hematoxylin and eosin-stained sections. In special situations, tissues may be e m b e d d e d in plastic so that electron microscopy can be p e r f o r m e d . Direct electronmicroscopy can also be p e r f o r m e d on intestinal contents or tissue homogenates. In situ DNA hybridization techniques on paraffine m b e d d e d tissues are available for certain viruses, such as Pacheco's herpesvirus, adenovirus, avian polyomavirus, psittacine circovirus, and paramyxovirus. I m m u n o h i s t o c h e m i c a l stains
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c a n d e t e c t c e r t a i n a n t i g e n s f r o m bacteria, f u n g i , viruses, a n d parasites a n d these t e c h n i q u e s c a n also b e utilized to i d e n t i f y s o m e cell m a r k e r s i n the diagnosis o f t u m o r s . T o x i c o l o g i c testing r e q u i r e s s o m e i d e a o f what t o x i n is b e i n g c o n s i d e r e d . This i n f o r m a t i o n o f t e n c o m e s f r o m the history a n d h i s t o p a t h o logic findings. C o n t a c t i n g the toxicology laboratory is essential for s u b m i s s i o n of the m o s t app r o p r i a t e tissues a n d a m o u n t s . T h e m o s t comm o n toxins tested are heavy metals, such as lead a n d zinc. Usually liver a n d k i d n e y are r e q u i r e d for this analysis, a l t h o u g h zinc a c c u m u l a t e s i n the p a n c r e a s preferentially. P o i s o n o u s p l a n t s c a n b e f o u n d i n the digestive tract a n d s u b m i t ted to a b o t a n i s t or university b o t a n y d e p a r t m e n t for i d e n t i f i c a t i o n . P o l y t e t r a f l u o r e t h y l e n e (Teflon) a n d o t h e r toxic i n h a l a t i o n p r o d u c t s are rarely d e t e c t a b l e i n tissues, a n d the diagnosis is usually m a d e with history o f e x p o s u r e , the presence of p u l m o n a r y e d e m a and hemorrhage, a n d e x c l u s i o n o f o t h e r causes o f death. Mycotoxins m a y be i m p l i c a t e d i n the case o f m u l t i p l e birds suffering liver d a m a g e . Aflatoxins c a n be detected i n foodstuffs, b u t usually by the time c h r o n i c liver d a m a g e is evident, the o f f e n d i n g f o o d s t u f f is o f t e n n o l o n g e r available. I n the case of a c u t e toxicosis, freeze samples of tile feed a l o n g with liver a n d kidney, p e n d i n g f u r t h e r investigation. I n c o n c l u s i o n , gross n e c r o p s y a n d p o s t m o r t e m d i a g n o s t i c testing is a very i m p o r t a n t p a r t o f avian m e d i c i n e , r e q u i r i n g a systematic a p p r o a c h to the e x a m i n a t i o n o f o r g a n s a n d the c o l l e c t i o n o f samples. D e v e l o p i n g a r e l a t i o n s h i p with a vete r i n a r y p a t h o l o g i s t is necessary to s u b m i t the a p p r o p r i a t e s a m p l e s a n d to o p t i m i z e the c h a n c e s for a r r i v i n g at a diagnosis.
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Recommended Reading 1. Altman RB, Forbes NA: Self-AssessmentColor Review of Avian Medicine. Ames, LA, Iowa State University Press, 1998 2. Campbell TW: Avian Hematology and Cytology (ed 2). Ames, IA, Iowa State University Press, 1995 3. Dorrestein GM: Diagnostic necropsy and pathology, in Altman RF, Clubb SL, Dorrestein GM, Quesenberry K (eds): Avian Medicine and Surgery. Philadelphia, PA, Saunders, 1997, pp 158-169 4. Fudge AM: Laboratory Medicine: Avian and Exotic Pets. Philadelphia, PA, Saunders, 2000 5. Graham DL: Necropsy techniques for pet birds, in 1986 Scientific Proceedings, 54th Annual Meeting of the American Animal Hospital Association. Denver, CO, American Animal Hospital Association, 1987, pp 455 6. Latimer KS, Rakich PM: Necropsy examination, in Ritchie BW, Harrison GJ, Harrison LR (eds): Avian Medicine: Principles and Application. Lake Worth, FL, Wingers Publishing, 1994, pp 355-379 7. Lowenstine LJ: The postmortem examination, in Proceedings of the Parrot Biology Symposium, Psittacine Research Project. Davis, CA, 1992, pp 37-42 8. Randall CJ: A Colour Atlas of Diseases of the Domestic Fowl and Turkey. Ames, IA, Iowa State University Press, 1985 9. Randall CJ, Reece RL: Color Atlas of Avian Histopathology. London, Mosby-Wolfe, 1996 10. Reavill D, Schmidt RE, Garner M: How to get the most for your pathology dollar. Exotic DVM 1(6):33-40, 2000 11. Reavill D: Getting an answer: tips on how to improve diagnostic testing, in Proceedings of the Annual Conference of the Association of Avian Veterinarians. Boca Raton, FL, Association of Avian Veterinarians, 2002, pp 271-278 12. Riddell C: Avian Histopathology (ed 2). Kennett Square, PA, American Association of Avian Pathologists, 1996 13. Ritchie BW: AvianViruses: Function and Control. Lake Worth, FL, Wingers Publishing, 1995 14. Samonr J: Avian Medicine. London, Mosby-Harcourt Publishers, 2000 15. Schmidt RE: 'Sudden' death in pet birds, in Proceedings of the Annual Conference of the Association of Avian Veterinarians. Boca Raton, FL Association of Avian Veterinarians, 1995, pp 473-478