AFIP Wednesday Slide Conference - No. 13
December 1, 1999

Conference Moderator:
LTC Jeff Eggers, Diplomate, ACVP
Chief Veterinary Pathology, CID
59th MDW/HSR
1255 Wilford Hall Loop, Bldg 4430
Lackland AFB, TX 78236-5319
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Case I - UFSM # 1 (AFIP 2559049)
Signalment: 4-year-old zebu cross, female.
History: This animal is from a group of 19 young adult cows that were in a pasture heavily infested with Tetrapterys multiglandulosa. The owner was advised of this being a poisonous plant, but still chose to leave the animals in the pasture. After 40 days he noticed that 4 cows were sick. There was subcutaneous dependant edema (submaxilar, ventral neck, and brisket), depression, and the animals tired easily. After 15 days, the cows were removed from the pasture but even then the clinical signs became progressively worse and the 4 cows died. Subcutaneous edema extended up to the ventral abdomen (kodakchrome A); the jugular vein was distended and pulsating and there was cardiac arrhythmia. After exercise the cows became breathless.
Gross Pathology: An extensive translucent gelatinous dependent subcutaneous edema was confirmed at necropsy. Marked cavitary edema (ascites, hydropericardium, and hydrothorax) and edema of the mesentery were also noticed. Discolored pale areas were seen in the myocardium through the epicardium and sharp white patchy areas and streaks (kodakcrome B) were present on the cut surface of the myocardium. The liver was slightly swollen and had a nutmeg appearance particularly on the cut surface.
Contributor's Diagnoses and Comments: Morphologic diagnosis:
Myocardium, necrosis of myofibers and fibrosis, multifocal, severe.
Etiologic diagnosis: Toxic cardiomyopathy
Etiology: Tetrapterys multiglandulosa toxin
Other histopathological changes included centrilobular congestion and fibrosis (congestive heart failure) of the liver.
The ingestion of plants of the genus Tetrapeterys, family Malpighiaceae, causes primarily degenerative and fibrotic changes in the myocardium, and related signs of cardiac failure. The natural disease occurs only in cattle over 1 year of age in southeastern Brazil and only in pastures where Tetrapterys spp. occur and after the cattle have stayed for 1 to 2 months on the pasture; it was experimentally reproduced in cattle by feeding fresh or dried sprouts of T. acutifolia and T. multiglandulosa (3). Most of the cases have subacute (1-5 weeks) or chronic (months) courses. Most common clinical signs include: edema of the brisket, prominent pulsating jugular vein and cardiac arrhythmia; in the terminal stages dyspnea and labored breathing are noticed. The natural disease occurs throughout the year with no seasonal preference, has variable morbidity rates, and high mortality. Occasionally some animals may recover; pregnant cows may abort. The main necropsy findings include: subcutaneous and cavitary edema; pale areas in the heart, perceptible through the epicardium; and sharp whitish areas and streaks across most of the cut surface of the myocardium.

When present, liver lesions are mild and related to congestive heart failure (nutmeg liver). The main histopathological findings are degeneration and lysis of myofibers marked massive necrosis of myocardium and fibrosis. Fibrosis occurs with a diffuse interstitial distribution or as extensive focal areas. Tetrapterys spp. poisoning must be differentiated from other primary or secondary cardiomyophies of cattle (1) such as high altitude (brisket) disease, St. George disease, and another disease with similar clinical signs (swollen brisket disease) (2) which occurs in the south of Brazil. These 3 diseases can be differentiated by epidemiological data. The first two are not reported in Brazil. Brisket disease presents changes (medial thickening) in the arteries and arterioles of the lungs and is usually reported on cattle above 2,000m while Tetrapterys spp. poisoning occur in cattle pasturing at 20-700m and only where Tetapterys spp. can be found. Other cardiomiopathies such as those induced by ionophore antibiotic (eg. monensin, salinomycin, lasalocid) poisoning should be included in the differential diagnosis.
Case 13-1. Antemortem photo. There is a pendulous and multinodular enlargement of the brisket area. (Image provided by Colegio Brasileiro de Patologia Animal, Seropedica, RJ, Brazil, with permission).
Case 13-1. Cardiac muscle. There are two focally extensive pale, white, areas in the left ventricular wall. (Image provided by Colegio Brasileiro de Patologia Animal, Seropedica, RJ, Brazil, with permission).
AFIP Diagnoses:
1. Heart, cardiomyocytes: Vacuolar degeneration, diffuse, moderate, with multifocal necrosis, fibrosis, mineralization, and chronic inflammation, zebu cross, bovine.
2. Heart, cardiomyocytes: Sarcocysts, few.
Conference Note: Although the specific etiology of this case is not found outside of southern Brazil, lesions of myocardial degeneration, necrosis and fibrosis occur in a variety of conditions including Vitamin E/Selenium deficiency, and various toxicities including those caused by gossypol, monensin and other ionophore antibiotics, Cassia occidentalis (coffee senna), Eupatorium rugosum (white snake root), Karwinskia humboldtiana (coyotillo), fluoroacetate containing plants (Acacia georginea, Gastrobilium spp, Oxylobium spp.), and others. Other causes of similar lesion include uremic cardiomyopathy and ischemia. Storage diseases such as generalized glycogenosis due to deficiency of 1,4-glucosidase may cause diffuse cardiomyocyte vacuolization.
Contributor: Universidade Federal de Santa Maria, Departamento de Patologia, 97119-900, Santa Maria, RS, Brazil
1. Peixoto PV, Loretti AP, Tokarnia CH: "Doenca do Peito inchado", Tetrapterys spp. poisoning, brisket disease and St. George disease: A comparative study. Pesq Vet Bras 15:43-50, 1995
2. Robinson WF, Maxie MG: The Cardiovascular System In: Pathology of Domestic Animals, 4th ed., vol. 3, eds. Jubb KVF, Kennedy PC, Palmer NC, pp. 27-31. Academic Press, San Diego, CA, 1993
3. Tokarnia CH, Gava A, Peixoto PV, Stolf L, Moaraes, SS: A "doenca do Peito inchado" (edema da regiao esternal) em bovinos no estado de Santa Catarina. Pesq. Vet. Bras. 9:73-83, 1989
4. Tokarnia CH, Peixoto PV, Doebereiner J, Consorte LB: Gava bovinos caracterizadas por alteracoes cardiacas. Pesq Vet Bras 9:23-44, 1989
Case II - OL 8257 (AFIP 2694777)
Signalment: 48-week-old, male, Charles River CD rat.
History: The rat was part of an experimental study. It was administered a test compound for four days. On the fourth day, the rat was found dead and was necropsied.
Gross Pathology: Kidneys were moderately enlarged and pale with many pinpoint white spots on the capsular surface and many white streaks on the cut surface, apparently following nephrons.

Contributor's Diagnoses and Comments:
1. Kidney: Severe, diffuse, acute, tubular degeneration and necrosis with intratubular, birefringent, acicular crystals.
2. Kidney: Mild, diffuse chronic progressive nephropathy ("old rat nephropathy").
Etiology: xemilofiban

Pathogenesis: Administration of large quantities of xemilofiban, forming crystalline precipitates in renal tubules and leading to obstruction of urine flow and tubule degeneration and necrosis.
Note: To preserve the water soluble crystals, a modified hematoxylin and eosin stain was performed, involving less time in hematoxylin and eosin and minimal washing steps.
Diffusely throughout the section, proximal tubules are mildly to moderately dilated. There is marked, diffuse degeneration and necrosis of proximal tubular epithelial cells with accumulation of small amounts of cellular debris in dilated tubules. Scattered tubules are lined by denuded basement membranes, which are occasionally partly mineralized. In scattered tubules, there are variable quantities of birefringent, refractile, acicular, 10-50 microns long, clear to brown crystals. Occasional crystals are present in the center of small aggregates of inflammatory cells, consisting of a mixture of macrophages, lymphocytes, plasma cells, and neutrophils. Most glomerular urinary spaces are mildly to moderately dilated. In addition, there are scattered regenerative ("basophilic") tubules and a few scattered small interstitial foci of lymphocytes, plasma cells, and macrophages. Glomerular tufts have segmental thickening of glomerular basement membranes and mesangial matrix with mild to moderate segmental thickening of Bowman's capsule with mild to moderate hyperplasia and hypertrophy of parietal cells.
The compound administered to the rat is a fiban known as xemilofiban, the development of which is currently discontinued. This compound is an ester prodrug, which is absorbed after oral administration and is de-esterified to its pharmacologically active acid.
Infrared spectroscopy, Ramen spectroscopy, time-of-flight secondary ion mass spectroscopy (TOF-SIMS), and liquid chromatography/mass spectroscopy (LC/MS) were used to identify the crystals in situ. These techniques confirmed that the intratubular crystals were composed of the acid metabolite of xemilofiban.
The fibans are a group of compounds that block the glycoprotein IIb-IIIa receptor on platelets. This receptor ordinarily binds fibrinogen, the final step of platelet aggregation, allowing platelets to aggregate in vivo and in vitro. The fibans reversibly block the receptor in a concentration-dependent way, causing a titratable inhibition of platelet aggregation.

Platelets of humans and dogs are highly sensitive to the effects of fibans. In dog toxicity studies, the limiting toxicological findings were intramural hemorrhages in the stomach and urinary bladder and/or gingival bleeding. The fibans evaluated did not cause obstructive nephropathy in dogs. However, rat platelets are comparatively insensitive to the fibans. Therefore, higher dosages are needed to produce toxicity in rats, and compound-related bleeding does not occur. The principal toxicity of these compounds in rats, at large multiples of human dosages, was the formation of crystalline precipitates in kidneys. These precipitates lead to obstruction of urine flow, varying degrees of renal inflammation, tubular degeneration, and secondary uremia.

Differential diagnosis for crystalline precipitates in renal tubules includes uric acid, sulfonamide, acyclovir, methotrexate, and oxalate crystals. Presence of uric acid crystals in renal tubules results from the destruction of cells, especially erythrocytes in newborn animals. Collecting tubules are filled with yellow crystals and their presence may give a radial pattern to yellowish streaks. Uric acid crystals may also appear in gout (especially in birds), where they may be found in the interstitial stroma with a radial pattern.

Various drugs, such as acyclovir, methotrexate, primadone, and sulfonamides, can cause acute renal failure through the deposition of crystalline material in renal tubules. Incidence of intraluminal sulfonamide crystals was greater when only less-soluble forms of the drug (sulfapyridine, sulfathiazole, and sulfadiazine) were available. This problem is currently rare, since newer sulfonamides are short acting and have greater solubility. The incidence of sulfonamide crystal precipitates is exacerbated by limited intake of water and acidic urine. The typical lesion is a plugging of the lower collecting tubules with masses of fine crystals, resulting in obstruction. The lesions are due to local toxic and mechanical effects. Hypersensitivity does not play as important a role in animals as it does in humans. The crystals of most of the sulfonamides are elongated, acicular, anisotropic, and yellowish in color.
Oxalate crystals in renal tubules can occur from ingesting plants with toxic levels of oxalate, ethylene glycol, oxalate salts, or plants infected with fungi that produce oxalates. Plants are the usual source of oxalate poison in sheep and cattle. Some of the common plants that contain oxalates are rhubarb (Rheum rhaponticum), halogeton (Halogeton glomeratus) and greasewood (Sacobatus vermiculatus), common sorrel (Rumex acetosa), pigweed (Amaranthus retroflexus), mangles, and sugar beets. Ethylene glycol is the usual source of oxalate poisoning in humans, dogs, and cats. It is metabolized in the liver by alcohol dehydrogenase, where it is oxidized to glycolic acid, then to glyoxylate, and finally to oxalate. Calcium oxalate is precipitated in renal tubules during the process of clearance. The crystals are nearly transparent, birefringent, irregularly rhomboidal, and 30-40 microns long.
AFIP Diagnoses:
1. Kidney: Tubular ectasia, diffuse, moderate, with intratubular birefringent acicular crystals, multifocal tubular degeneration and necrosis, and subacute tubulointerstitial nephritis, Charles River CD rat, rodent.
2. Kidney: Nephropathy, mild, characterized by glomerular and tubular basement membrane thickening, chronic interstitial nephritis, and tubular regeneration (chronic nephropathy of rats).
Conference Note: The contributor has provided an excellent discussion of both the pathogenesis of fiban toxicity and the differential diagnosis for crystalline precipitates in renal tubules. Conference participants agreed with the contributor's morphologic diagnosis. A discussion of the differential diagnosis and pathogenesis of nephrosis ensued. This was followed by a brief review of the characteristic features of chronic progressive nephropathy in rats ("old rat nephropathy"). These features include interstitial fibrosis, chronic inflammation, tubular ectasia and proteinosis, glomerular basement membrane thickening and mesangial proliferation. Participants remarked that this case provides an excellent example of a more acute lesion superimposed on more chronic lesions.
Contributor: Searle/Monsanto, 4901 Searle Parkway, Skokie, IL 60077
1. Jones TC, Hunt RD, King NW: Veterinary Pathology, 6th ed., pp. 1131-1132, pp. 724-726. Williams & Wilkins, Baltimore, MD, 1997
2. Maxie GM: The Urinary System. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 490-493. Academic Press, San Diego, CA, 1993
3. Levin S, Friedman RM, Cortez E, Hribar J, Nichols M, Schlessinger S, Fouant M, Khan N: Lesion and identification of crystalline precipitates of glycoprotein IIb-IIa antagonists in the rat kidney. Tox Pathol 27:38-43, 1999
4. Perazella, Mark MD: Crystal-induced acute renal failure. Am Journ of Med 106:459-465, 1999
Case III - 99T6-20 (AFIP 269553)
Signalment: Young adult, Xenopus laevis (African clawed frog), female
History: Several frogs had been found dead. Others appeared to have increased shedding and external mucus production. Aeromonas hydrophilia was isolated from external lesions. Ova from frogs are used for calcium channel studies.
Gross Pathology: Skin is rough and reddish over abdomen and legs. Frogs do not change pigment/coloration in response to light.
Laboratory Results: Aeromonas hydrophilia pure culture
Contributor's Diagnosis and Comments: Skin: Dermatitis, subacute, multifocal with nematodes consistent with Pseudocapillaroides (Capillaria) xenopi.
These laboratory-raised frogs are housed in a state-of-the-art aquarium system that incorporates filtration and water changes. They had been at the institution for 8 months. Frogs from the same tank necropsied 2 months before had dermatitis but no evidence of nematodiasis. Tank treatment with levamisole (2 times, 2 weeks apart) resulted in no further evidence of infection.
AFIP Diagnosis: Skin: Hyperplasia, epithelial, diffuse, moderate, with mild chronic-active dermatitis and many intraepithelial aphasmid nematodes, African clawed frog (Xenopus laevis), amphibian.

Conference Note: Conference participants unanimously agreed with the contributor's identification of Capillaria xenopodis (Pseudocapillariodes xenopi). It is the only nematode parasite reported in the epidermis of African clawed frogs. The key histologic features of this aphasmid parasite include: stichosomes (large round basophilic structures) thin cuticle, thin to inapparent musculature, and digestive tract lined by many uninucleate cells.

Only a few affected animals in the population show the clinical signs of rough skin, anorexia and emaciation. Histologically, there is epidermal hyperplasia and mild inflammation. This epidermal hyperplasia significantly limits the normal epidermal functions of respiration and osmotic balance. Generally animals waste away for 2-3 months and eventually succumb to bacterial septicemia, most commonly caused by Aeromonas hydrophila.
Other cutaneous pathogens of frogs include Citrobacter freundii, Flavobacterium, chytridiomycosis, chromomycosis, Chlamydia, and Mycobacterium.
Other nematode parasites that localize in epithelium include: Trichosomoides crasicauda in the urothelium of rats, Gongylonema sp. in various locations including the esophagus of cattle and primates, Anatrichosoma sp. in the nasal mucosa of primates, and Capillaria bohmi in the nasal mucosa of foxes.
There are many other significant species of Capillaria. C. hepaticum infects the hepatic parenchyma of rodents and rarely dogs. C. aerophilus infects the nasal passages of many animals. C. feliscati infects the urinary tract of cats. C. micronata infects the urinary tract of minks. C. putorii infects the stomach of cats.

Contributor: Air Force Research Laboratory, 2509 Kennedy Circle, Brooks AFB, TX 78235-5118
1. Crawshaw GJ: Amphibian Medicine. In: Kirk: Current Veterinary Therapy XI Small Animal Practices, pp. 1219-1230. WB Saunders, Philadelphia, PA, 1992
2. Barker IK, Dreumel AAV, Palmer N: The Alimentary System. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer NC, 4th ed., vol. 2, p. 269. Academic Press, San Diego, CA, 1993
3. Dungworth DL: The Respiratory System. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer NC, 4th ed., vol. 2, p. 562. Academic Press, San Diego, CA, 1993
3. Kelly R: The Liver and Biliary System In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer NC, 4th ed., vol. 2, p. 376. Academic Press, San Diego, CA, 1993
4. Maxie MG, Prescott JF: The Urinary System In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer NC, 4th ed., vol. 2, p. 518. Academic Press, San Diego, CA, 1993
5. Ruble G, Berzins IK, Huso DL: Diagnostic Exercise: Anorexia, Wasting, and Death in South African Clawed Frogs. Lab Ani Sci, 45(5):592-594, 1995
Case IV - 99250 (AFIP 2699550)
Signalment: 2-year-old, male New Zealand white rabbit, Oryctolagus cuniculus.
History: This animal received an intradermal injection along the dorsal midline of 20.0 mg purified brown recluse spider toxin (venom) as part of a spider bite therapy study. The submitted sections of skin were taken 48 hrs post-injection.
Gross Pathology: A 6 cm X 5 cm irregularly shaped, pale blue to blue-black mottled area of skin was present on the dorsal midline. Mild erythema and dermal and subcutaneous edema extended ventrally from the central lesion.

Contributor's Diagnosis and Comments: Haired skin and subcutis: Necrosis and hemorrhage, focally extensive, severe with necrotizing vasculitis, fibrin thrombi, collagen degeneration and edema.

The lesion is typical of "necrotic arachnidism" associated with the bite of the North American brown recluse spider (Loxosceles reclusa). Bites from brown recluse spiders are common and show considerable variation in clinical presentation. The early lesion is characterized by erythema, pruritus, pain, swelling and blister formation. In more severe cases, localized necrosis develops with a central area of deep blue to purple discoloration surrounded by a red rim of erythema. The lesion spreads in a gravity-dependent manner and eventually progresses to form a disfiguring dark black eschar.
Brown recluse venom is cytotoxic and hemolytic. Several enzymes have been identified in the venom including lipases, hyaluronidase, alkaline phosphatase, and most importantly sphingomyelinases. Sphingomyelinases are primarily responsible for both tissue necrosis and complement-dependent hemolysis. The pathogenesis is thought to involve intravascular coagulation locally at the site of the bite followed by necrosis and an influx of PMNs. Endothelial damage and thrombosis of small capillaries have been demonstrated ultrastructurally as early as 3 hours after envenomation.
More recent studies have shown Loxosceles venom induces E-selectin expression on endothelial cells and stimulates endothelial production of NF-[Kappa]B-dependent proinflammatory chemokines IL-8, GM-CSF, and GRO [Alpha]. IL-8 is a potent neutrophil agonist, and parenteral infusion of IL-8 neutralizing antibody has been shown to attenuate, but not eliminate lesion development experimentally. Leukocyte-derived inflammatory mediators likely play an important role in the continued progression and refractory nature of these lesions.
Histologic evaluation of chronic lesions in people reveals persistent arterial wall necrosis, subcutaneous fat necrosis and residual scarring of the dermis and subcutis.
In addition to local tissue necrosis, systemic signs are occasionally reported following brown recluse spider bite, most commonly headache, joint pain, abdominal pain and vomiting. "Systemic loxoscelism" refers to a rare, severe systemic reaction usually occurring several days after envenomation. This reaction is characterized by massive hemolysis, acute renal failure, thrombocytopenia, and can progress to disseminated intravascular coagulation, coma and death, particularly in young children.
AFIP Diagnosis: Haired skin and subcutis: Necrosis and hemorrhage, focally extensive, severe, with necrotizing vasculitis, fibrin thrombi, acute inflammation, collagen degeneration and edema, New Zealand white rabbit, lagomorph.
Conference Note: The differential diagnosis discussed in conference for this type of lesion included thermal injury, bacterial septicemia, drug reaction, type three hypersensitivity (Arthus) reaction to bacterial toxins or drugs, and infectious agents such as Rickettsia rickettsi and several toxins.
Contributor: Pathobiology, Clinical Research, Wilford Hall Medical Center, 1255 Wilford Hall, Lackland AFB, TX 78236
1. Butz WC, Stacy LD, Heryford NN: Arachnidism in rabbits: Necrotic lesions due to the brown recluse spider. Arch Path, 9: 97-99. 1971
2. Berger RS, Adelstein EH, Anderson PC: Intravascular coagulation: The cause of necrotic arachnidism. J of Invest Derm, 61:142-150, 1973
3. Clowers TA. Wound assessment of the Loxosceles reclusa spider bite. Journal of Emergency Nursing, 22:283-287, 1996
4. Masters EJ. Loxoscelism: Images in clinical medicine. NEJM, 339(6):379, 1998
5. Pucevich MV, McChesney T. Histopathologic analysis of human bites by the brown recluse spider. Arch Dermatol, 119: 851, 1983
6. Rees RS, O'Leary JP, King LE. The pathogenesis of systemic loxoscelism following brown recluse spider bite. J Surg Res. 35(1), 1983
7. Tambourgi DV, et al. Sphingomyelinases in the venom of the spider Loxoxceles intermedia are responsible for both dermonecrosis and complement-dependent hemolysis. Biochem and Biophys Res Commun 251(1):366-73, 1998
8. Wilson DC, King LE. Spiders and spider bites. Derm Clini 8(2): 277-286, 1990
9. Wasserman GS, Anderson PC. Loxoscelism and necrotic arachnidism. J. Toxicol Clin Toxicol 21(4&5): 451-472, 1983-84
J Scot Estep, DVM
Captain, VC, USA
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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