AFIP Wednesday Slide Conference - No. 15
January 5, 2000

Conference Moderators:
Dr Richard J Montali and Dr James T Raymond
Department of Pathology
National Zoological Park
Washington, DC 20008
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Case I - PV98-1227 (AFIP 2693098)
Signalment: 2½-year-old male chimpanzee (Pan troglodytes)
History: There was a short course of CNS disease that abruptly ended a budding movie career. Other chimpanzees in the gang colony had vague signs of a "cold". This sub-adult was the only one to develop loss of balance and difficult prehension with arm pain. There was opisthotonus described but normal cranial nerve reflexes. The lumbar CSF was clear. Multiple fluffy, cloud-like areas in the MRI were interpreted as the density of hemorrhages.
Gross Pathology: The brain was mildly swollen in the calvarium. As depicted in the representative 2x2 slide of formalin-fixed sections, there were many (>10) random areas of soft purple discoloration, both in the cortices and brain stem. These spanned gyri and were easily depressed. Some were partially liquefied with petechiae and ecchymoses rimming foci in the basal ganglia and thalamus. There was brown mucus in a bronchus, suggesting aspiration with subpleural purple foci, but no palpable consolidation.
Case 15-1. Multifocally within the cerebral cortex and brain stem there are areas of brownish-pink discoloration.
Laboratory Results: Blood cultures negative. The WBC rose during the week from 23,000 to 35,800. There was mild anemia. Blood lead levels were normal.
Contributor's Diagnosis and Comments: Multifocal necrotizing encephalitis, granulomatous, severe, with numerous amoebae.

Etiology: Balamuthia mandrillaris

Sections from all levels of the brain revealed variably severe to massive malacia and intense inflammation with macrophages, giant cells and often large numbers of neutrophils. There was lymphocytic perivascular cuffing with some thrombi and neuronal necrosis, as well as some foci of parenchymal hemorrhage. The corpus striatum was especially severely affected with destruction of the putamen.
Numerous amebic trophozoites are present in the brain but are often difficult to visualize with H&E, unless the hematoxylin is well developed. The organisms prove negative with Giemsa, PAS and silver stains. Mucicarmine was not attempted but is reported to stain trophozoites.
The diagnosis was confirmed by immunohistochemistry, which proved positive for Balamuthia and negative for Naegleria and Acanthamoeba.

There was mild concurrent interstitial pneumonitis and the immunostains did decorate a few organisms in the septa. This is consistent with the belief that this pathogen can enter the respiratory system from inhaled dusts as well as the more usually discussed rhinocerebral route. Balamuthia is said to be ubiquitous in soil but has never been isolated from the environment. There is reported predilection for Old World primates.
AFIP Diagnosis: Brain: Encephalitis, necrotizing, subacute, multifocal, severe, with necrotizing vasculitis and amoebae, chimpanzee (Pan troglodytes), nonhuman primate.
Conference Notes: Balamuthia mandrillaris, Acanthamoeba sp., and Naegleria fowleri are free-living amoebae that have been reported to cause fatal encephalitis/meningoencephalitis in humans and animals. B. mandrillaris has been reported in old world monkeys, sheep, humans and a horse.

In tissues, Balamuthia mandrillaris and Acanthamoeba spp. are nearly identical. Both appear in two forms: 15-30mm diameter, round to irregular trophozoites, and rare 10-15mm diameter cysts. Trophozoites and cysts are scattered individually and in clusters throughout the neuropil and perivascular spaces. Despite their similar light microscopic appearance, the two organisms can be tentatively differentiated by the presence of multiple nucleoli in B. mandrillaris trophozoites, as opposed to the single nucleolus of Acanthamoeba spp. trophozoites.
Contributor: PATHVET Consultation Services, 3015 Roxanne Avenue, Long Beach, CA 90808
1. Lozano-Alarcon F, Bradley GA, Houser BS, Visvesvara GS. Primary Amoebic Meningoencephalitis due to Naegleria fowleri in a South American Tapir. Vet Pathol 34(3):239-243, 1997
2. Kinde H, Visvesvara GS, Barr BC, Nordhausen RW, Chiu PHW. Amebic Meningoencephalitis caused by Balamuthia mandrillaris (leptomyxid ameba) in a horse. J Vet Diagn Invest 10(4):378-381, 1998
3. Rideout BA, Gardiner CH, Stalis IH, Zuba JR, Hadfield T, Visvesvara GS. Fatal Infections with Balamuthia mandrillaris (a Free-Living Ameba) in Gorillas and Other Old World Primates. Vet Pathol 34(1):15-22, 1997
Case II - E98-478-2 (AFIP 2686003)
Signalment: Cheetah (Acinonyx jubatus)
Contributor's Diagnosis and Comments: Idiopathic necrotizing leukoencephalopathy-leukodystrophy
White matter in the corona radiata, internal capsule, and centrum semiovale is diffusely widened, pale, and crossed by numerous prominent branching capillaries lined by plump endothelium. The sheathed axons are also widely separated and the myelin sheaths are frequently fragmented with the presence of numerous small round eosinophilic balls. Dispersed throughout the white matter are a moderate number of foamy gitter cells that occasionally contain phagocytosed eosinophilic debris. Scattered pyknotic nuclear debris and a small number of mineralized spherules are also present. Numerous reactive astrocytes with large round nuclei and abundant homogenous eosinophilic cytoplasm, form a spiderweb of interconnected cell processes. Occasionally, these reactive astrocytes have vacuolated cytoplasm. The grey matter is unaffected except for a thin rim adjacent to the white matter. Within this zone of grey matter are a small number of reactive astrocytes and scattered eosinophilic balls. Occasional vessels are surrounded by perivascular cuffs composed primarily of lymphocytes along with rare hemosiderin laden macrophages.
Demyelinating diseases include 1) dysmyelination (genetic disorders of myelin formation); 2) demyelination secondary to neuronal destruction (neuronolytic demyelination or Wallerian degeneration secondary to viral infection or toxicity); and 3) primary demyelination (diseases where demyelination is the sole disease process). The CNS lesions in these cheetahs are characterized by extensive demyelination with at least some degree of concurrent axonal degeneration. The etiology is uncertain. Transmissible spongiform encephalopathy has been described in four cheetahs that were born in captivity in Great Britain. Histopathology reported in those cases included widespread axonal degeneration and demyelination of all spinal cord tracts that extended up the pyramidal tracts in the medulla and as far cranially as the internal capsule. However, varying degrees of spongiosis was also seen in grey matter and a few vacuoles were observed within the perikarya of some neurons.
Through July 1, 1999, a total of 30 confirmed cases of leukoencephalopathy have been documented in cheetahs. Clinically, affected cheetahs have evidence of progressive loss of vision, have difficulty prehending food, and are uncoordinated. All cases have marked reactive astrocytosis predominantly in the cerebral cortical white matter with degeneration and necrosis. Lesions seem to begin in the corona radiata as a reactive astrocytosis that evolves into demyelination, axonal loss, leukoencephalomalacia, and cavitation in the oldest lesions. The lesions have remarkable bilateral symmetry with Wallerian degeneration in descending proprioceptive pathways, crus cerebri, longitudinal fibers of the pons, the pyramids, the decussation, and the lateral corticospinal tracts.
Reportedly, the problem first appeared in March, 1997, affects only older cheetahs, is not familial, has emerged in multiple facilities in the United States, and has been confirmed in a single case in England. The same signs and lesions have been observed in two Florida panthers necropsied in 1994 and 1997. Both of these animals were at least 8 years old (A. De lahunta, personal communication). Antemortem diagnosis can be made by either CT or MR imaging but MRI is the most reliable procedure. Attempts to identify a possible viral etiology, mycotoxin, Vitamin B deficiency, or reactions to vaccines or medications are ongoing (Dr. Linda Munson, UC, Davis).
AFIP Diagnosis: Brain: Leukoencephalopathy characterized by necrosis, gemistocytic astrocytosis, numerous gitter cells, mineralization and lymphoplasmacytic inflammation, cheetah (Acinonyx jubatus), feline.
Conference Notes: Several diseases that are unusual in most mammals commonly affect captive cheetahs. For example, gastritis associated with Helicobacter-like organisms, veno-occlusive disease and glomerulosclerosis have been found to be prevalent in captive cheetahs in facilities in the United States and the Republic of South Africa.
Cheetahs are remarkable in their lack of genetic diversity. It has been hypothesized that a severe population crash might explain the genetic uniformity of the species. Calculations based on diversity of mitochondrial DNA and hypervariable minisatellite loci suggest that the population bottleneck occurred about 10,000 years ago, near the end of the last ice age, in the late Pleistocene. At that time, extinction of a number of large vertebrates occurred on several continents. The cheetah's lack of genetic diversity may play a role in its susceptibility to unusual diseases.
Contributor: College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401
1. Allen IV: Demyelinating diseases. In: Greenfield's Neuropathology, eds. Adams JH, Corselleis JAN, Duchen LW, 4th ed., John Wiley & Sons, pp. 338-384. New York, NY, 1984
2. Baron T, Belli P, Madec JY, Moutou F, Vitaud C, Savey M: Spongiform encephalopathy in an imported cheetah in France. Vet Record 141(11):270-271, 1997
3. Kirkwood JK, Cunningham AA: Epidemiological observations on spongiform encephalopathies in captive wild animals in the British Isles. Vet Record 135(13):296-303, 1994
4. Munson L, de Lahunta A, Citino S, Radcliffe R, Neiffer D, Montali R, Stalis I: Leukoencepahlopathy in cheetahs. Am Assoc Zoo Vet (abstract), 1999
5. Munson L, Nesbit JW, Meltzer DG, Colly LP, Bolton L, Kriek NP: Diseases of captive cheetahs (Acinonyx jubatus jubatus) in South Africa: a 20-year retrospective survey. J Zoo Wildl Med 30(3):342-347, 1999
6. Menotti-Raymond M, O'Brien SJ: Dating the genetic bottleneck of the African cheetah. Proc Natl Acad Sci USA 90(8):3172-3176, 1993
7. Peet RL, Curran JM: Spongiform encephalopathy in an imported cheetah (Acinonyx jubatus). Aust Vet J 69(7):171, 1992
Case III- P-081-96 (AFIP 2683470)
Signalment: Three-year-old male rock hyrax (Procavia capensis)
History: 5 months prior to death, the animal was noted to have some sneezing, coughing and slight weight loss. The animal's condition stabilized and the cough became intermittent. Immediately prior to death, the respiratory signs progressed. A left pulmonary lobectomy was done during an exploratory thoracotomy. The animal was electively euthanized the following day due to poor prognosis based on the results of histopathology.
Gross Pathology: At necropsy, the right lung contained multifocal to coalescing firm white glistening nodules affecting up to 60% of the pulmonary parenchyma. The left lung was removed during surgery 1 day prior to necropsy and had a similar appearance.
Laboratory Results: Cultures are positive for Mycobacterium bovis.
Histology: Section of lung with severe alterations of normal architecture. Few remnants of tissue identifying structures remain. The majority of the sections are effaced by variably sized 15 to 100 mm diameter nodules. The nodules are centrally composed of large numbers of whirling epithelioid cells with oval nuclei and moderate amounts of eosinophilic granular to vacuolated cytoplasm. Surrounding the nodules are low to moderate numbers of lymphocytes admixed with plasma cells, and spindle cells with moderate amounts of eosinophilic fibrillar cytoplasm (fibrous connective tissue). Multifocal bronchial lumens contains low numbers of macrophages admixed with lymphocytes and red blood cells. In one section multifocal bronchi are filled with massive numbers of mature and degenerate neutrophils admixed with fewer lymphocytes, plasma cells, macrophages, cellular debris and mucus. In less severely affected areas, alveolar lumens are filled with varying combinations of neutrophils, macrophages and large numbers of red blood cells admixed with homogeneous eosinophilic proteinaceous material (edema). Remaining pulmonary vessels are filled with moderate numbers of red blood cells (congestion).

Contributor's Diagnoses and Comments:
1. Bronchopneumonia, diffuse, granulomatous, massive with intralesional acid fast bacilli.
2. Tracheitis, diffuse, granulomatous, severe.
This hyrax died from a severe granulomatous pneumonia and disseminated granulomas caused by acid fast bacilli. Cultures of lung tissue isolated Mycobacterium bovis. Acid fast positive bacilli were found in trachea, lung, thyroid and spleen. In most sections concentrations of bacilli were low with the exception of the trachea, where bacilli were more numerous.
AFIP Diagnosis: Lung: Granulomas, epithelioid, coalescing, rock hyrax (Procavia capensis), cavid.
Conference notes: Mycobacterium spp. are nonmotile, nonspore forming, pleomorphic bacilli, they are weakly Gram positive and acid-fast positive. Three species of Mycobacterium are considered tubercle bacilli, M. tuberculosis, M. bovis, and M. avium. Though there is some species predilection with each, all three can infect a wide range of species, and especially immunocompromised animals.
Due to the presence of several compounds in their cell walls, mycobacteria are able to escape killing by phagocytic cells and induce delayed hypersensitivity. Cord factor is a surface glycolipid found in pathogenic mycobacteria; it stimulates granuloma formation. Sulfatides are sulfur-containing glycolipids that prevent fusion of phagosomes of macrophages with lysosomes. Another cell wall constituent, lipoarabinomannan (LAM), is a heteropolysaccharide that inhibits macrophage activation by interferon-gamma and induces macrophages to secrete TNF-a and IL-10, which suppresses mycobacteria-induced T-cell proliferation.
The mycobacteria are initially able to replicate in naive macrophages. After a few weeks, however, T cell mediated immunity develops (delayed hypersensitivity). CD4+ helper T cells activate macrophages by secreting interferon-gamma, enabling the macrophages to kill the bacilli via the release of reactive nitrogen intermediates. CD8+ suppressor T cells kill macrophages that harbor mycobacteria, causing caseous necrosis; the mycobacteria cannot grow in the acidic, extracellular environment of the caseous core of a granuloma. The classic granuloma of tuberculosis is the result of this process.
Chronic infection frequently follows, with a balance between bacterial replication and destruction. Stress of any kind can tip the scale in the direction of the mycobacteria and produce fulminant infection.
Contributor: Department of Pathology, Wildlife Health Center / WCS, 185th St. and Southern Blvd. Bronx, New York, 10460
1. Hines ME, Kreeger JM, Herron AJ: Mycobacterial Infections of Animals: Pathology and Pathogenesis, Lab An Sci 45(4):334-347, 1995
2. Jackson R, Cooke MM, Coleman JD, Morris RS, de Lisle GW, Yates GF: Naturally occurring tuberculosis caused by Mycobacterium bovis in brushtail possums (Trichosurus vulpecula): III. Routes of transmission and excretion. NZ Vet J 43:322-327, 1995
3. Krebs JR, Anderson RM, Clutton-Brock T, Donnelly CA, frost S, Morrison WI, Woodroffe R, Young D: Badgers and bovine TB: Conflicts between conservation and health. Science 279:817-818, 1998
4. Miller J, Jenny A, Rhyan J, Saari D, Suarez D: Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms. J Vet Diag Lab Inv 9:244-249, 1997
5. Rhyan J, Saari D: A conparative study of the histopathologic features of bovine tuberculosis in cattle, fallow deer (Dama dama), Sika deer (Cervus nippon), red deer and elk (Ce-rvus elaphus). Vet Pathol 32:215-220, 1995
6. Samuelson J: Infectious diseases. In: Robbins Pathologic Basis of Disease, eds. Cotran RS, Kumar V, Collins T, 6th ed., pp. 349-352. WB Saunders, Philadelphia, 1999
Case IV - X6664 (AFIP 2695443)
Signalment: 8-year-old, female, captive red panda (Ailurus fulgens)
History: Traumatic event resulted in fracture of ulna and muscular damage around axilla. The red panda died 3 days later.
Contributor's Diagnosis and Comments: Stomach: Erosions and ulcers, acute, multifocal, with fibrinous thrombi
Etiology: Associated with physiological stress
Histologically, there is multifocal degeneration and acute coagulative necrosis and loss of gastric mucous neck cells, chief cells, and parietal cells. There are superficial erosions of the gastric mucosa that sometimes extend deep to the vicinity of the muscularis mucosa. Multifocally, there is brown, granular to globular pigment along the base and around few distended veins in some of the eroded areas. At the base of the mucosa, there is venous distention with margination of neutrophils and, in some areas, infiltration of the eroded mucosa by few neutrophils. Few ectatic veins within the lamina propria contain intravascular fibrin thrombi. In few sections of stomach, there is ulceration of the mucosa that is characterized by extension of the mucosal necrosis to the muscularis mucosa.
Generally, erosions and ulcers of this type are usually associated with physiological stress. The "stress ulcers" can occur multifocally throughout the gastric and sometimes duodenal mucosa. In humans, they have been associated with shock, sepsis, burns, trauma, and increased cranial pressure. The pathogenesis of stress ulcers is still not completely understood but possible mechanisms are decreased blood flow to the mucosa (ischemic necrosis), disruption of the gastric mucous layer, decreased bicarbonate buffer, increased acid secretion, and direct damage to the gastric mucosal epithelium.
In wild and captive nondomestic animals, stress erosions and ulcers are not uncommon. Spontaneous acute gastric erosions and ulcers due to physiological stress have been previously reported in captive vervet monkeys (Cercopithecus aethiops). In one study, stress ulcers occurred in approximately 1/3 of necropsied monkeys and were associated with individual housing conditions. Gastric ulcers have been reported in stranded marine mammals such as the sperm whale and were noted in sea lion pups affected by ecological disruptions associated with El Nino. Llamas hospitalized for extended periods of time have been known to develop third compartment ulcers. In the case of this red panda, the stress associated with the traumatic injuries most likely caused the gastric erosions and ulcers.
AFIP Diagnosis: Stomach, mucosa: Erosions and necrosis, multifocal, acute, with fibrin thrombi, red panda (Ailurus fulgens), procyonid.
Conference Notes: Gastric erosions and ulcers are common in most domestic and exotic species and have been associated with many etiologies, including stress, septicemia, uremia, disseminated intravascular coagulation, glucocorticoid usage, and administration of non-steroidal anti-inflammatory drugs. In dogs, gastric ulcers are occasionally associated with gastrin-secreting pancreatic tumors and mast cell tumors. Ulcers in the pars esophagea region of pigs are associated with the practice of feeding finely ground feed.

Gastric erosions are characterized by loss of the superficial epithelium that produces a defect that does not cross the muscularis mucosae. Frequently there is an associated acute inflammatory infiltrate and extrusion of a fibrin-containing purulent exudate into the lumen. Gastric ulcer is defined as a breach of the mucosa that extends through the muscularis mucosa into the submucosa or deeper.
Contributor: Department of Pathology, National Zoological Park, Washington, DC 20008
1. Crawford JM: The Gastrointestinal Tract. In: Pathologic Basis of Disease, eds. Cotran RS, Kumar V, Collins T, 6th edition, WB Saunders Company, Philadelphia, PA, 1999
2. Jauniaux T, Brosens L, Jacquinet E, Lambrights D, Addink M, Smeenk C, and Coignoul F: Postmortem investigations on winter stranded sperm whales from the coasts of Belgium and The Netherlands. J Wildl Dis 34: 99-109, 1998
3. Mbaruk AS, Tarara RP, Else JG, Sayer PD: Spontaneous acute gastric mucosal erosions and ulcerations in vervet monkeys. J Zoo Wildl Med 26: 67-71, 1995
4. Spraker TR, Gulland F, DeLong R: The impact of El Nino on marine mammals. Proc Am Assoc Zoo Vet: 160-161, 1998
5. Smith BB, Pearson EG, Timm KI: Third compartment ulcers in the llama. Vet Clin North Am Food Anim Pract 10: 319-30, 1994
6. Tarara MA, Tarara RP, Suleman MA: Stress-induced gastric ulcers in vervet monkeys: The influence of life history factors. J Zoo Wildl Med 26: 72-75, 1995
J Scot Estep, DVM
Captain, United States Army
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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