C O N F E R E N C E 10 9 November 2016

Joint Pathology Center Veterinary Pathology Services

WEDNESDAY SLIDE CONFERENCE 2016-2017

C o n f e r e n c e 10 9 November 2016

CASE I: AzVDL 02-3362 (JPC 4019839). Cytologic Description: The preparation has large areas of dense cellularity. There are Signalment: Nine-year-old, female, numerous linear structures (nematode Siamese cross, (Felis catus). uterus) containing oval bioperculated ova are present within the dense areas. Well- History: The cat presented for chronic preserved and degenerate parasite ova are cystitis unresponsive to antibiotic therapy also scattered throughout the background. and dietary changes. Pneumocystogram The ova are approximately 60 microns in revealed a thickened uneven bladder wall length with a granular interior and a thick with a small lumen. The cat was referred to refractile shell. In the thinner areas of large an internal medicine service with the cellular densities, the nucleated cells are primary differential diagnosis of neoplasia. present in organized sheets with well- Ultrasound showed an irregular mass in the defined cytoplasmic borders. The cells have bladder wall at the trigone. A “traumatic round nuclei with mild anisokaryosis and a catheterization” of the bladder was scant to small amount of basophilic performed and cytologic preparations were cytoplasm. The background has a large submitted for evaluation. number of lysed and degenerate cells with fewer small clusters of intact epithelial cells Gross Pathology: None. showing the same morphology as those within the dense clusters. There are areas of Laboratory results: The CBC at monolayer adjacent to the dense clusters presentation to the specialty service was with a moderate number of neutrophils and within reference interval. The only eosinophils. Eosinophils, neutrophils, and abnormality in the chemistry profile was small lymphocytes are found throughout the hyperglycemia. The abnormalities in the background. Occasional small lymphocytes urinalysis included glucosuria, mild pyuria contain a few eosinophilic to azurophilic and large numbers of variably sized trans- granules. itional epithelial cells.

Table 1: Capillarid Species Name Location Host Eucoleus bouhmi frontal sinus fox, dog Eucoleus aerophilus bronchi dog, cat, fox Aonchotheca putorii stomach, intestine bear, hedgehog, raccoon, swine, bobcats, mustelids, cat

Aonchotheca spp intestine ruminants Calodium hepaticum liver rodents, many occasional hosts including humans.

Pearsonema plica urinary bladder dog, fox, wolf Pearsonema feliscati urinary bladder cat

Contributor’s Cytologic Diagnosis: nematodes have been placed in several new Transitionalcell hyperplasia with mild genera based on location within the host, the suppurative and eosinophilic inflammation most accepted of which are Eucoleus (nasal and fragmented Pearsonema (Capillaria) sinus and bronchi), Aonchotheca (intestine) feliscati nematode and numerous bio- and Pearsonema (urinary bladder) (Table perculated eggs. 2)2. Pearsonema plica and P. feliscati, infect the domestic dog, fox, wolf, and cat. Contributor’s Comment: Urinary nema- In most cases, the nematode is loosely todiasis is caused by several genera and attached to the urinary bladder mucosa and, affects numerous species of domestic and less often, ureteral mucosa with mild wild animals (Table 1).2, 5,10 The nematode inflammation and edema evident on species found in the kidney, ureters or renal histologic examination of the affected vasculature are associated with clinical tissue.7,8 Clinical disease is uncommon but disease. The most spectacular of which is has been reported in the dog and fox and the giant kidney worm of dogs, Dio- rarely in the cat.7,8 The prevalence of these ctophyme renale, that eventually destroys parasites in the dog and cat is not known. and replaces the renal parenchyma. However, a prevalence of 76% and 59% has Nematodes of the genera Pearsonema been reported in two dog breeding kennels (Capillaria), on the other hand, are found in the United States.7 In this heavily within the urinary bladder and are often an parasitized population, hematuria, dysuria incidental finding with minimal clinical and pollakiuria were common findings in the disease evident.2,10 The capillarid dogs with confirmed infection.

are elongate and approximately 60 microns in length and 27 microns in width. They have bipolar plugs (bioperculate), a thick shell with “globular” ridges enclosing a single cell.10 The literature does not provide morphologic or biological

Urinary bladder, cat. Several large aggregates of protein encase tangential sections of adult characteristics other nematodes. (green arrows.) (Wrights, 16X) than the host to distinguish P. plica In the cat, the disease is rarely reported in and P. feliscati. The cat is included in host the United States;6 however, in Australia, an species for both P. feliscati and P. plica in incidence of greater than 30% was found in Georgis’ Parasitology for Veterinarians 8 with no mention of differential one survey study. No evidence of clinical 2 cystitis was seen in the infected cats. On characteristics. The capillarid in this case is histological examination of the urinary given as P. feliscati based on host. bladder in the infected cats, the nematodes were superficially embedded in the mucosa with no breaching of the basal layer or basement membrane. A moderate JPC Cytologic Interpretation: Presence of inflammatory infiltrate that included eosino- Peasonema spp nematode fragments and phils was seen in association with the ova, mild transitional cell dysplasia, and low embedded parasite. The lifecycle for P. grade eosinophilic and neutrophilic feliscati is not determined.2,6 It is assumed inflammation, Siamese cross, Felis catus. to be similar to P. plica which is thought to be through the earthworm as an intermediate Conference Comment: The contributor host or a transport host such as a bird. Adult provides a compelling cytologic specimen worms are 2.5 to 5 cm in length. The ova and outstanding review of capillarid Table 2: Urinary Nematodes Name Host Dioctophyme renale dog, mink

Pearsonema plica, P. feliscati dog, fox, wolf, cat

Stephanurus dentatus swine Trichosomoides crassicauda rats Crassicauda boopis whales

Urinary bladder, cat. Tangential sections of the adult female parasites contina numerous 25x50um eggs within the uterus. (Wrights, 124X).

nematodes affecting a wide variety of large numbers of eggs were found in all veterinary species. As mentioned by the bladders containing five or more adults.9 In contributor, the Capillaria genera have been this case, the mild to moderate eosinophilic reclassified based on the location of the and neutrophilic inflammation may be adult parasite in the host; however, the secondary to a high parasite burden. genus name Capillaria is still widely used in the veterinary literature. In both dogs and cats, Peasonema plica has been reported in the urinary bladder and Typically, infection with Pearsonema ureter submucosa and is typically associated feliscati is of little pathologic significance to with mild subclinical inflammation and the cat and is usually considered an edema.1,7 There is a higher prevalence of the incidental finding. Most studies show no parasite in wild red foxes (Vulpes vulpes) in relationship between the presence of adult many European countries and it is associated capillarids in the bladder and significant with an increased pathogenicity. This can cystitis.8,9 Histologically, Pearsonema result in severe cystitis, pollakiuria, dysuria feliscati resides in the superficial epithelium and hematuria in this species.1,3 In this case, of the urinary bladder and does not penetrate both Pearsonema plica and Pearsonema the basement membrane.5 However, if the feliscati have been implicated in urinary ureters become occluded with adult bladder infection in cats, conference nematodes, cats may display the clinical participants could not distinguish between signs of post-renal obstruction. The highest two cytologically. reported number of adult worms present from a single urinary bladder was 25 and

nematode can be more difficult to appreciate than on histologic tissue section. However, the presence of the highly characteristic ova confirms the presence of a urinary capillarid nematode.

Participants also noted vacuolation, binucleation, anisokaryosis, and prominent nucleoli within the reactive sloughed urothelium. Transitional epithelial cells are among the most pleomorphic cells in the body and can demonstrate marked reactive change in response to a variety of insults. For this reason, the conference moderator Urinary bladder, cat. Eggs are 25x50um, brown, with a bioperculated (arrows) brown refractile shell and reminded participants that care must be granular contents. (Wrights, 830X) taken prior to over-interpreting reactive urothelium as malignancy. Conference participants readily identified numerous bioperculate eggs free within the Contributing Institution: sample and inside the reproductive tract of Arizona Veterinary Diagnostic Laboratory adult nematode fragments. Pearsonema spp 2831 N. Freeway are a subclassification of aphasmid Tucson, AZ 85705 nematodes of the family Trichuridae. http://cals.arizona.edu/vdl/ Histologically, aphasmid nematodes are characterized by thin eosinophilic cuticle, References: reduced polymyarian- coelomyarian musculature, two hypodermal 1. Basso W, Spanhauer Z, Arnold D, bacillary bands, a stichosome esophagus, Deplazes P. Capillaria plica (syn. 4 a spiny sheath and oval bioperculate eggs. Pearsonema plica) infection in a dog Cytologically, specific features of the adult with chronic pollakiuria: Challenges in the diagnosis and treatment. Parasitol Int. 2014; 63:140142. 2. Bowen DD. Georgis’ Parasitology for Veterinarians. 2008. (Kindle Locations 9850-9856). Elsevier Health. Kindle Edition. 3. Fernandez-Aguilar X, et al. Pearsonema (syn Capillaria) plica associated cystitis in a Fennoscandian artic fox (Vulpes lagopus): A case report. Acta Vet Scand. 2010; 52:39. 4. Gardiner CH, Poynton SL. Urinary bladder, cat. There are numerous clusters of Aphasmids. In: Gardiner CH, transitional epithelial cells with \in the specimen. Poynton SL, eds. An Atlas of (Wrights, 523X) Metazoan Parasites in Animal Tissues. Washington, DC: Armed

Forces Institute of Pathology; absence of hepatocellular cytomegaly or 1999:40-43. karyomegaly. 5. Lambertsen RH. Diseases of the common fin whale (Balaenoptera The patient re-presented in January of 2015 physalus): Crassicaudiosis of the for increasing lethargy, dull mentation and urinary system. Journal of waxing and waning appetite. The horse was Mammology. 1986; 67(2):353-366. treated with minocycline, metronidazole, 6. Lautenslager JP. Internal helminthes pentoxifylline, lactulose, vitamin E, and of cats. Vet Clin North Amer. 1976; prednisolone. 6(3):353365 7. Senior DF, Solomon GB, The patient presented again in February of Goldschmidt MH, et al. Capillaria 2015 due to progression of clinical signs and plica infection in dogs. J Am Vet failure to respond to treatment. Euthanasia Med Assoc. 1980;176(9):901-905. was elected. 8. Waddell AH. Further observations of Capillaria feliscati in the cat. Aust Gross Pathology: At necropsy, the liver Vet J. 1968;44(1):33-34. was markedly reduced in size weighing 3.1 9. Wilson-Hanson S, Prescott CW. kg (0.7% of body weight) and had thick Capillaria in the bladder of the rounded margins. Some of the lobes domestic cat. Aust Vet J. 1982; appeared multinodular and there were 59:190-191. multifocal areas of capsular fibrosis 10. Zajac, Anne M.; Conboy, Gary A. Veterinary Clinical Parasitology. Laboratory results: 2011. (Kindle Locations 3752-3763). February 2015: (reference ranges) John Wiley and Sons. Kindle GGT 294 IU/L (8-22) Edition. SDH 16 IU/L (0-8) Resting bile acids 36 uMOL/L (4-11.5) Plasma ammonia 242 UG/DL (5-59)

Postmortem liver heavy metal screen CASE II: 15N0368 (JPC 4084209). Iron 5100 ppm (100-300)

Signalment: Ten-year-old, mare, Histopathologic Description: Liver: Within quarterhorse, (Equus ferus caballus). all sections, the hepatic capsule is variably thickened, and the lobular architecture is History: This animal presented to the pronounced due to marked portal to portal teaching hospital in November of 2014 for bridging fibrosis. Dissecting bands of weight loss. At that time, blood work fibrosis variably infiltrate the surrounding revealed hyperproteinemia and elevated parenchyma causing isolation of hepatic blood ammonia (see laboratory results lobules and smaller clusters of hepatocytes. below). Ultrasound of the liver revealed Rare isolated hepatocytes appear hyper- rounded margins. A liver biopsy was eosinophilic with pyknotic to karyolytic performed and revealed nodular nuclei (individual cell apoptosis). Diffusely, regeneration, bridging fibrosis, and arteriolar hepatocytes contain variable amounts of and bile duct proliferation. The clinical coarse, dark brown, granular pigment suspicion at that time was chronic (hemosiderin). Kupffer cells and pyrrolizidine alkaloid toxicity despite an

The liver was markedly reduced in size weighing 3.1 kg (0.7% of body weight) and had thick rounded margins. Some of the lobes appeared multinodular and there were multifocal areas of capsular fibrosis (Photo courtesy of: VMTH Anatomic Pathology, 1 Garrod Drive, University of California, Davis, CA 95616 http://www.vetmed.ucdavis.edu/vmth/lab_services/anatomic_pathology/index.cfm)

macrophages, which course throughout affected sections, there are frequent, areas of fibrosis, contain similar, but more irregular, refractile crystals associated with variably sized, dark brown cytoplasmic small numbers of multinucleated giant cells granules (hemosiderophages). Areas of and epithelioid macrophages (foreign body fibrosis also contain small to moderate response). numbers of lymphocytes and plasma cells, with rare neutrophils, and abundant variably Contributor’s Morphologic Diagnosis: sized bile ducts and small-caliber blood Liver: Severe, chronic, bridging portal and vessels. Occasional small aggregates of capsular fibrosis with lobular collapse, veno- lymphocytes and plasma cells are observed occlusion, biliary hyperplasia and marked throughout the parenchyma. In some areas, hepatocellular hemosiderosis. the hepatic parenchyma has been completely replaced by mature fibrous tissue and Contributor’s Comment: The abundant proliferating bile ducts. In addition, mature hepatocellular and Kupffer cell pigment was fibrous tissue occasionally appears to confirmed as iron using Perls’ iron stain. occlude central and portal veins with rare Heavy metal analysis of the liver revealed evidence of recanalization (venoocclusive an iron concentration of 5100 ppm, far disease). Within one section there are large, exceeding the normal upper limit of 300 multifocal areas of acute hemorrhage and ppm. Perl’s staining also revealed iron edema that cause separation and dis- deposits within smooth muscle trabeculae of organization of the hepatic cords. the spleen, and within renal tubular Occasional individual collagen bundles are epithelium at the corticomedullary junction. segmentally darkly amphophilic (sidero- Iron within splenic trabeculae and within calcinosis). Within the most severely fibrous tissue in the liver is occasionally

transferrin and the transferrin receptor.9 Secondary hemochromatosis occurs from excessive intestinal absorption of iron either due to iron excess within the diet, or as a response to increased demand for erythro- poiesis. Secondary hemochromatosis can occur with any condition leading to chronic hemolysis. Although a very small amount of iron is eliminated through the bile, the body has no natural way of responding to excess iron and therefore iron accumulates over time, primarily in the liver. Iron Liver, horse. There is moderate bridging portal fibrosis which breaches the limiting plate and entraps hepatocytes accumulation results in hepatocellular (center). Hepatocytes, especially in portal areas, contain toxicity through production of free radicals abundant intracytoplasmic iron. (HE, 168X) and organelle dysfunction, including lysosomal injury.8 This can lead to deposited in conjunction with calcium salts hepatocellular necrosis that progresses to (siderocalcinosis). In addition, the most bridging fibrosis, bile duct hyperplasia, and severely affected sections shows deposition venoocclusive disease, as observed in this of refractile clear crystals that are associated case. In humans, hemochromatosis can also with a granulomatous foreign body increase the risk of hepatocellular neoplasia9 response. These crystals are not birefringent and in birds, has been shown to predispose under polarized light. to certain bacterial infections such as Yersinia pseudotuberculosis.4 With the exception of certain avian species, including mynahs and toucans, hemo- Distinguishing between primary and chromatosis is a rare condition in domestic secondary iron storage disease can be species. A hereditary form of especially difficult in chronic cases. In hemochromatosis has been reported in Salers and Salers-cross cattle,8 and there have been individual case reports in horses.6 Cases of dietary iron overload have also been reported in sheep and cattle. Iron storage disease is classically divided into two entities: hemosiderosis (iron overload in the absence of clinical signs) and hemochromatosis (iron overload leading to hepatic damage including fibrosis, inflammation, and liver failure). In human medicine, iron storage disease is additionally subdivided into primary and secondary categories. Primary iron storage disease involves an inherent abnormality in iron Liver, horse. The liver stains deep blue with a Perl’s iron stain. (Perl’s iron, 5X) (Photo courtesy of: VMTH metabolism. In humans, this is most Anatomic Pathology, 1 Garrod Drive, University of commonly the result of one of two missense California, Davis, CA 95616 mutations in the HFE gene which encodes a http://www.vetmed.ucdavis.edu/vmth/lab_services/anatom ic_pathology/index.cfm) protein involved in the interaction between

humans, the pattern of hemosiderin enterocytes absorb approximately 1 to 2 mg deposition can be helpful.6 In primary iron of iron per day from the diet via divalent storage disease, hemosiderin accumulates metal transporter-1 (DMT-1) and a heme first in hepatocytes while in secondary carrier protein-1 (HCP-1) on the luminal hemosiderosis, iron accumulates first within surface of the enterocytes.5,9 Iron is Kupffer cells and macrophages (reticulo- transported from the cytoplasm of the endothelial system). However, this requires enterocyte to the circulation by that the liver is examined early in disease ferroportin.1,7 Absorbed iron circulates progression. In this case, a primary bound to transferrin and is used primarily by abnormality in iron metabolism is suspected erythroid precursors in the synthesis of based on the lack of clinical or histologic heme. Macrophages in the spleen clear dead evidence of a second underlying disease and dying erythrocytes and release the iron process leading to chronic hemolysis, and from heme to export it to the circulation or feeding of a standard equine diet. store it in ferritin. In iron-overload, transferrin is quickly saturated and iron is JPC Morphologic Diagnosis: Liver: stored in the liver and various other tissues Fibrosis, portal and bridging, diffuse, due to the lack of a regulated pathway for marked with hepatocellular degeneration effective iron excretion.3 and loss and intra-hepatocellular hemosiderosis, Quarterhorse, Equus ferus As mentioned above, hepatocytes are a caballus. major site of iron storage as ferritin and are also responsible for the production of type II Conference Comment: The contributor acute phase protein, hepcidin, in response to provides an outstanding example and inflammatory cytokine, interleukin-6.1,3,5,7 thorough review of hemochromatosis in Hepcidin is transported in by blood by humans and veterinary species. Free iron is alpha-2-macroglubulin and blocks the highly toxic to tissues due to its ability to release of iron from enterocytes and participate in the generation of hydroxyl radical formation via the Fenton or Haber- Weiss reaction with hydrogen peroxide (H2O2) leading to lipid peroxidation and DNA damage.5 As a result, iron is typically bound to transferrin while in circulation and either ferritin or hemosiderin when stored and sequestered in tissue. When iron is bound to these proteins, it cannot participate in these injurious reactions. Ferritin concentration is highest in the liver, spleen, and bone marrow and is stored in hepatocytes and/or macrophages.1,3,5,7 Liver, horse. Occasionally, fibrosis of portal areas In hepatocytes, iron is derived from plasma obliterates portal veins (veno-occlusive disease). (HE, transferrin, while iron stored within 100X) (Photo courtesy of: VMTH Anatomic Pathology, 1 Garrod Drive, University of California, Davis, CA 95616 macrophages is a result of erythrocyte http://www.vetmed.ucdavis.edu/vmth/lab_services/anatom breakdown. Normally, to offset the ic_pathology/index.cfm) attritional loss of daily iron, duodenal

findings in this case. Elevated sorbital dehydrogenase (SHD: 16 IU/L [0-8]) and elevated plasma ammonia (242 UG/dL [5- 59]) indicate hepatocellular injury and decreased hepatic function respectively. In addition, elevated gamma-glutamyl trans- peptidase (GGT: 294 IU/L [8-22]) and elevated resting bile acids (36 uMOL/L [0- 20]) indicate cholestasis and biliary hyperplasia with decreased hepatobiliary function.1,2,5,8

Contributing Institution: Liver, horse: Multifocally, non-refractile crystals are University of California Davis present within aggregates of epithelioid macrophages and VMTH Anatomic Pathology multinucleated giant cells. (HE, 400X) (Photo courtesy 1 Garrod Dr. of: VMTH Anatomic Pathology, 1 Garrod Drive, University of California, Davis, CA 95616 Davis, CA 95616 http://www.vetmed.ucdavis.edu/vmth/lab_services/anatom http://www.vetmed.ucdavis.edu/vmth/lab_se ic_pathology/index.cfm) rvices/anatomic_pathology/index.cfm macrophages by degrading the iron exporter, Clin Path Data: ferroportin. In humans, decreased hepcidin See charts appended at end of document. synthesis caused by mutations in the hepcidin gene, HAMP, causes severe References: hemochromatosis in juveniles.1,5 1. Bain PJ. Liver. In: Latimer KS ed. Within the cytoplasm, iron is stored as Duncan and Prasse’s Veterinary Laboratory Medicine Clinical ferritin, which is reconverted into iron as th needed by the body. If tissue ferritin levels Pathology. 5 ed. Ames, IA:Wiley- are high, ferritin aggregates into hemo- Blackwell; 2011:213-224. siderin globules, which is much more 2. Brockus CW. Erythrocytes. In: difficult to revert back to free iron. In Latimer KS ed. Duncan and Prasse’s Veterinary Laboratory Medicine hepatocytes overwhelmed with iron, most th iron is stored as hemosiderin. Ferritin and Clinical Pathology. 5 ed. Ames, hemosiderin readily stain with Pearls IA:Wiley-Blackwell; 2011:4 Prussian blue, demonstrated nicely in this 3. Cullen JM, Stalker MJ. Liver and case.1,5 biliary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Iron is a direct hepatotoxin and iron Pathology of Domestic Animals. Vol overload often results in the formation 2. 6th ed. Philadelphia, PA:Elsevier; periportal bridging fibrosis with little 2016:272. inflammation.1,5 In addition to the markedly 4. Galosi L, Farneti S, Rossi G, et al. elevated postmortem liver heavy metal Yersinia pseudotuberculosis, screen (Iron: 5100 ppm [100-300]), clinical serogroup O:1A, infection in two pathology data from the provided serum amazon parrots (Amazona aestiva biochemistry supports the histologic and Amazona oratrix) with hepatic

hemosiderosis. J Zoo Wildl Med. accidents. Otherwise, she was clinically 2015; 46(3):588-591. healthy. Initial blood work showed azotemia 5. Kumar V, Abbas AK, Fausto N. Red (BUN: 26 mg/dL [reference range: 5-20]; blood cell and bleeding disorders. In: serum creatinine: 2.0 mg/dL [reference Robbins and Cotran Pathologic range: 0.6-1.6]) and glucosuria (4+) with Basis of Disease. 9th ed. normal blood glucose. There was also Philadelphia, PA:Elsevier Saunders; increased ALT (458 IU/L [reference range: 2015:650-651. 10-55]). Her urine was isosthenuric and 6. Lavoie JP, Tuescher JP. Massive contained fine granular casts. Her blood iron overload and liver fibrosis pressure was normal (systemic blood resembling haemochromatosis in a pressure: 140 mmHg). There was no history racing pony. Equine vet J. of exposure to toxins. Subsequent blood 1993;25(6):552-554 work one month later showed increased 7. Mazzaro LM, Johnson SP, Fair PA, azotemia (BUN: 23 mg/dL; serum Bossart G, Carlin KP, Jensen ED, creatinine: 3.0 mg/dL), ALT (707 IU/L), and Smith CR, Andrews GA, Chavey PS, mild proteinuria (UPC: 1.3 [normal <0.5]). Venn-Watson S. Iron indices in bottlenose dolphins (Tursiops Gross Pathology: A wedge biopsy of the truncatus). Comp Med. 2012; renal cortex was submitted for evaluation. 62:508-515. Wedge biopsies of liver were also harvested 8. O’Toole D, Kelly EJ, McAllister and submitted to a different diagnostic MM, et al. Hepatic failure and laboratory. No gross abnormalities were hemochromatosis of Salers and observed at surgery. Salers-cross cattle. Vet Pathol. 2001; 38(4):372-389. Laboratory results: 9. Pietrangelo A. Hereditary The unfixed renal tissue that had been hemochromatosis—A new look at an submitted for IF evaluation, and a portion of old disease. N Engl J Med. 2004; the liver sample were submitted to Colorado 350(23):2383-2397. State Diagnostic Laboratory to measure 10. Weiss DJ. Iron and copper copper. There was evidence of copper deficiencies and disorders of iron hepatopathy (copper 2,690 ppm [>1,500 metabolism. In: Weiss DJ, Wardrop ppm in the liver is considered toxic]) and KJ, eds. Schalm’s Veterinary copper in the kidney 243.00 ppm (relevant Hematology. 6th ed. Ames, reference range >100 ppm in non-hepatic IA:Wiley-Blackwell; 2010:170. tissue being indicative of toxicity). Urine was also submitted approximately 10 days after the biopsy. SDS-PAGE analysis of urine demonstrated proteinuria due to the presence of low molecular weight proteins, consistent with tubular damage and absence CASE III: B110209 (JPC 4086860). of glomerular injury. An aliquot of this urine sample was submitted to PennGen Signalment: Eight-year-old, female, which documented generalized amino- Labrador retriever, (Canis familiaris). aciduria and glucosuria without ketonuria or cystinuria warranting a diagnosis of History: The dog had a history of polyuria acquired Fanconi syndrome. and polydipsia with several urinary

glomeruli have minimal to mild mesangial

Kidney, dog. At left, tubular epithelium is swollen by clear vacuoles (hydropic degeneration) and numerous brown to pink intracytoplasmic granules. There are few necrotic and sloughed cells within the obliterated lumen. At right, atrophic and ectatic tubules are surrounded by collagen (arrows) which is infiltrated by low numbers of lymphocytes and neutrophils. (HE, 224X)

Histopathologic Description: (H&E): expansion and the interstitium is mildly There is loss of the apical brush border of expanded by fibroplasia with scattered the proximal tubules. Scattered tubules are aggregates of lymphocytes and necrotic with cellular casts within lumens macrophages. and attenuated or absent epithelial lining. Tubules are dilated and undergoing Special stains: Rhodanine stain: Scattered degeneration with single cell necrosis and tubular epithelial cells contain small and apoptosis. The tubular epithelial cells large red-brown granules, consistent with contain variably sized cytoplasmic vacuoles copper. PAS demonstrates multifocal with abundant granular pigment. There is marked loss of the apical brush borders. marked epithelial cell karyomegaly. A few The trichrome stain demonstrates a few

12 large regions of mild to moderate interstitial fibrosis.

Contributor’s Morphologic Diagnosis: Moderate to severe tubular degeneration with necrosis, regeneration, atrophy, epithelial cell karyomegaly and scattered intracytoplasmic copper within tubular epithelial cells. Mild multifocal interstitial fibrosis.

Contributor’s Comment: The lesions presented in this case are indicative of renal proximal tubular injury, which was Kidney, dog. Glomeruli are mildly enlarged, hypercellular and have increased basement membrane clinically supported by the urinalysis results. material. Bowman’s capsule is also expanded. (HE, These acquired lesions have been associated 256X) with copper storage hepatopathy as a part of Wilson’s disease in humans and dogs. normal dogs. In contrast, acquired proximal Acquired Fanconi syndrome is characterized renal tubulopathies have been loosely by impaired reabsorptive function of the characterized in the literature and is proximal renal tubules. Clinical features attributed to many causes including copper include excessive loss of water, glucose, hepatopathy, leptospirosis, hypo- amino acids, uric acid in the urine, and parathyroidism, ethylene glycol toxicity, electrolyte abnormalities. The inherited form antibiotic, and chicken jerky treats. of Fanconi-like syndrome is well described in Basenji dog and is thought to be due to In humans, Wilson’s disease is an autosomal increased amounts of cholesterol in the recessive disorder of copper metabolism caused by mutations in the ATP7B gene. Decreased expression of this gene leads to decreased biliary excretion of copper resulting in hepatic copper accumulation. Patients with Wilson’s disease also accumulate copper in various tissues including the brain, eye, and kidney. Copper storage diseases have been reported in several canine breeds including Bedlington terrier, Labrador retriever, Doberman pinscher, Dalmatian, Skye terrier and West Highland white terrier. The inherited form of copper storage disease has been well documented in Bedlington terrier in which the copper metabolism domain containing 1 (COMMD1) gene is affected. The Kidney, dog: Tubules are occasionally lined by COMMD1 protein is important for copper regenerating epithelium with large nuclei and basophilic cytoplasm. (HE, 256X) excretion into bile during states of elevated intracellular copper. Hepatic histopathology tubular epithelial brush border compared to of copper associated hepatopathies generally

13 present with mixed inflammation (neutrophilic, lymphoplasmacytic, histiocytic) and is usually localized to the centrilobular regions. Centrilobular necrosis, bridging fibrosis, and cirrhosis have also been described in copper-associated hepatitis. Of note, chronic liver injury will lead to increased amounts of copper in the hepatocytes, sometimes making it difficult to discern whether intracellular copper is the cause or the effect of the liver injury.

A few case series/reports have documented acquired proximal renal tubulopathies Kidney, dog. A Masson’s trichrome demonstrates the large amount of fibrous connective within the associated with copper storage hepatopathy interstitium, which is difficult to visualize on the HE- in dogs. Breeds included are: Clumber stained section. (Masson’s trichrome, 88X) spaniel, West Highland white terrier, Cardigan Welsh corgi, and Labrador low copper diet can also contribute to retriever. The renal histologic findings in improvement of clinical signs. previous reported cases were consistent with the case presented here. Histologic JPC Morphologic Diagnosis: Kidney, evaluation of the renal tissues showed tubules: Epithelial degeneration, re- proximal tubular epithelial degeneration, generation, and necrosis, diffuse, marked, necrosis, and regeneration. The tubular with karyomegaly, few tubular casts, epithelial cells were plump with variably intracytoplasmic pigment and interstitial sized vacuoles. Using rhodanine stain, one fibrosis, Labrador retriever, Canis case reported copper deposition was mainly familiaris. localized to the corticomedullary junction and medullary areas; however copper Conference Comment: The contributor staining can be variable. Therefore, mild provides an excellent example and thorough tubular epithelial cell degeneration and loss review of copper-associated acquired of the apical brush border in the setting of Fanconi-like syndrome. This syndrome is clinical symptoms of Fanconi syndrome characterized by polyuria, polydipsia, should alert the pathologist to possible hyposthenuria, glucosuria with normo- copper-mediated damage to the renal glycemia, hyperphosphaturia, proteinuria, proximal tubules. Assay of copper levels in and amino aciduria due to impaired renal the liver or kidney samples supports this tubular absorption of glucose, phosphates, pathogenesis. sodium, potassium, uric acid, and amino 1,2,4,7 acids. In this case, this animal had In previous case reports of copper glucosuria with normoglycemia and amino- hepatopathy induced proximal renal tubular aciduria indicating poor proximal disease, there was improvement and convoluted tubular functioning. Glucose is resolution of clinical signs with copper normally resorbed in the renal proximal chelation therapy, specifically using d- tubules via the sodium-glucose co-transport 1,7,9 penicillamine. Additional therapies system. The concentration gradient including supportive care, antioxidants, and established by this system also promotes sodium resorption from the tubular fluid. In

are damaged or defective so low molecular weight proteins, like smaller globulins and some albumen, do not get resorbed from the ultra-filtrate and are excreted in the urine. Hemorrhagic/inflammatory proteinuria occurs due to hemorrhage or inflammation within the renal tubules, renal pelvis, or lower urinary tract. In this case, SDS-PAGE detected low molecular weight proteins within the urine and suggests that the primary lesion is in the proximal tubules rather than the glomeruli.8 This finding is confirmed by the histopathology of the Kidney, dog: A rhodanine stain demonstrates abundant copper within degenerate/necrotic tubules. (Rhodanine, kidney in this case. The characterization of 252X) proteinuria by SDS-PAGE is a useful antemortem clinical tool to identify the main congenital or acquired tubular defects of pathophysiologic mechanism involved. Fanconi-like syndrome, glucose is not resorbed and will cause an osmotic diuresis. Although not reported in this case, this This diuresis causes a marked decrease in animal was likely in a secretory metabolic kidney’s ability to concentrate urine and will acidosis due to renal tubular acidosis and increase urine volume; indicated by polyuria 1,7,9 loss of bicarbonate through the urine. In and isosthenuria reported in this case. cases of Fanconi-like syndrome, the renal The polydipsia is likely secondary to proximal tubules fail to resorb filtered compensation from increased fluid loss in bicarbonate.3 Other causes of secretory the urine. metabolic acidosis include vomiting of intestinal contents rich in bicarbonate, Conference participants discussed the diarrhea, and an inability to swallow saliva significance of the reported presence of low rich in bicarbonate in ruminants during molecular weight proteins in the urine using dysphagia.3 The hallmark of this type of sodium dodecyl sulfate polyacrylamide gel acidosis is concurrent hyperchloremia as the electrophoresis (SDS-PAGE). In general, body attempts to maintain electroneutrality. there are four main types of proteinuria: pre- In addition, the anion gap will typically be renal, glomerular, tubular, and normal because unmeasured anions are not hemorrhagic/inflammatory. In pre-renal increased.3 proteinuria, small proteins such as hemoglobin dimers, myoglobin, and light Contributing Institution: chains are present in the plasma at increased Department of Biosciences concentrations and pass through the The Ohio State University glomerulus and are incompletely resorbed 8 Columbus OH by fully functioning tubules. Glomerular https://vet.osu.edu/ proteinuria is characterized by damage to the glomerulus, thus enabling high molecular weight and negatively charged proteins to Clin Path Data: leak into the filtrate and pass into the urine 8 See chart appended at end of document. due to loss of selective permeability. In tubular proteinuria, proximal renal tubules

References: Veterinary Clinical Pathology. 2nd ed. Ames, IA: Blackwell Publishing; 1. Appleman EH, Cianciolo R, 2008:458-460. Mosenco AS, Bounds ME, et al. 9. Thompson MF, Fleeman LM, Transient Acquired Fanconi Kessell AE, Steenhard LA, Foster Syndrome Associated with Copper SF. Acquired proximal renal Storage Hepatopathy in 3 Dogs. J tubulopathy in dogs exposed to a Vet Intern Med. 2008; 22:1038-1042. common dried chicken treat: 2. Coronado VA, O’Neill B,Nanji M, retrospective study of 108 cases Cox DW. Polymorphisms in canine (2007-2009). 2013. Aust Vet J. ATP7B: candidate modifier of 9;368-73. copper toxicosis in the Bedlington terrier. Vet J. 2008; 2:293-96. 3. George JW, Zabolotsky SM. Water, electrolytes, and acid base. In: CASE IV: 16-2046 (JPC 4084201). Latimer KS, ed. Duncan and Prasse’s Veterinary Laboratory Signalment: Three-year-old, male, English Medicine Clinical Pathology. 5th ed. setter mix, (Canis familiaris). Ames, IA: Iowa State University Press; 2011:163-166. History: The three-year-old intact male 4. Hill TL, Breitschwerdt EB, Cecere English setter mix was picked up by county T, Vaden S. Concurrent Hepatic animal control when it was found roaming Copper Toxicosis and Fanconi’s freely in a small rural community in Syndrome in a Dog. J Vet Intern Southern Arizona. Aspirate cytology from Med. 2008; 22:219–22. the preputial mass and several cutaneous 5. Hoffman G. Copper-Associated masses were submitted. Liver Diseases. Vet Clin Small Anim. 2009; 39:489–511. Gross Pathology: The dog had a large 6. Hooper AN, Roberts BK. Fanconi ulcerated circumferential mass effacing the syndrome in four non-basenji dogs preputial mucosa and numerous variably exposed to chicken jerky treats. J Am sized cutaneous masses throughout the Anim Hosp Assoc. 2011; 47:e178-87. caudal dorsum, right and left flank, and 7. Langlois DK, Smedley RC, Schall inguinal regions. The superficial cervical WD, Kruger JM. Acquired proximal lymph nodes were enlarged. renal tubular dysfunction in 9 Labrador retrievers with copper- Laboratory results: associated hepatitis (2006-2012). J The dog was positive for Ehrlichia canis Vet Intern Med. 2013; 27:491-9. using in-house ELISA testing. 8. Stockham SL, Scott MA. Urinary system. In: Fundamentals of

(domestic dogs, coyotes, foxes, and wolves).3 The tumor has a global distribution and highest prevalence in regions with limited canine population control. The tumor spreads by sexual contact and is usually localized to the mucosal surface of the external genitalia of both male and female dogs. However, masses have been reported in the mucosal surfaces of the nasal passage, oral mucosa, anus, and conjunctival surfaces of the eye.3,7 Preputial mass, dog. The prepuce is markedly expanded Metastatic disease is not common but has by a large ulcerated neoplasm, in addition to other masses been documented. In most cases, metastasis on the caudal dorsum, flank, and inguinal regions. (Photo courtesy of: Arizona Veterinary Diagnostic is to the regional lymph nodes, but extension Laboratory, University of Arizona, of the neoplasm to skin, kidney, brain, bone, http://azvdl.arizona.edu/ peritoneum, and other tissues has been reported.1,3 The tumor cells are aneuploid Cytologic Description: The aspirate and have a unique long interspersed nuclear preparations from the dermal masses all element (LINE-1) that may be useful to revealed the same process. The preparations confirm TVT origin in cases involving have high cellularity with large numbers of tumors in unusual sites.10,12 intact cells for evaluation. There is minimal hemodilution. The nucleated cell population While TVT is a relatively uncommon tumor consists of round cells with moderate in most of the United States, rural areas that anisokaryosis and anisocytosis. The nuclei have higher numbers of intact dogs, such as are round with coarse “ropey” chromatin many parts of Southern Arizona, cases are and often have a single large pale lightly seen on a regular basis. The histological and basophilic nucleolus. The cytoplasm is cytological characteristics of the neoplastic lightly basophilic and contains small to cells along with the location of the mass in moderate numbers of discrete round vacuoles. Mitotic figures are common and atypical mitoses are seen. There are occasional neutrophils and small lympho- cyte seen intermixed with the neoplastic round cells.

The cytology preparations from the prepuce showed the same cytological findings.

Contributor’s Cytologic Diagnosis: Transmissible venereal tumor (TVT) metastatic to skin. Preputial mass, dog. The cytologic preparation is densely Contributor’s Comment: Transmissible cellular on a background of light blue protein. (Wright- venereal tumor is a transplantable neoplasm Giemsa, 100X) that affects members of the canid family

across several continents about 500 years ago and is currently found on every continent in the world other than Antarctica.5,12 It is the oldest known continuously passaged somatic cell lineage.5 As mentioned by the contributor, TVT is mainly transmitted during coitus, but can also be transmitted by licking, biting, or rubbing behaviors.2,8 The infecting cells are physically transplanted and grow as a xenograft in host tissue. Neoplastic cells are thought to be of histiocytic origin and are Preputial mass, dog. Neoplastic cells are monomorphic immune-positive for vimentin, lysozyme, with mild anisokaryosis and a few lymphocytes and alpha-1-antitypsin, glial fibrillary acidic neutrophils in the background. (Wright-Giemsa, 200X) protein, and are immune-negative for association with the external genitalia make cytokeratin, S100, and muscle markers.1,2,9 the diagnosis of this tumor relatively straight forward. In this case, the cytological and Typical gross findings of TVT include histological findings from the skin lesions solitary or multiple papillary, nodular, and the preputial lesion were identical ulcerated, and inflamed masses on the supporting metastatic disease. There is one mucous membranes of the penis, prepuce, report of a prepubertal female dog without vulva, and vagina, and/or skin and subcutis any genital involvement.4 It is proposed that of the head, neck, limbs, trunk, scrotum, and the tumor cells were transplanted from the perineum. There is occasional extension to dam by cohabitation and grooming/social the uterus and cervix.2,9 A recent study behaviors. While multicentric disease demonstrated ocular lesions as the single cannot be ruled out, it is unlikely that manifestation of TVT with extension to the multiple sites of transplantation through adjacent conjunctiva and nictitating intact haired skin would explain the membrane, presumably by extra-genital presence of the skin lesions in this adult male dog with a concurrent preputial mass.

JPC Cytologic Diagnosis: Fine needle aspirate, cutaneous mass: Transmissible venereal tumor, English setter mix, Canis familiaris.

Conference Comment: Canine transmissible venereal tumor (TVT), also known as Sticker tumor or venereal granuloma, is an extremely old and remarkably stable transmissible cancer that first arose in canids approximately 11,000 years ago.2,5 Based on Preputial mass, dog. Neoplastic mast cells have moderate amounts of light blue cytoplasm and discrete vacuoles. genetic studies, the founder breed is thought There are two mitotic figures in this field. (Wright- to be closely related to wolves or ancient Giemsa, 600X) Eastern Asian dog breeds.1 TVT dispersed

spread through biting. Tumors occur primarily in the mouth, head, and neck. DFTD cells evade immune destruction via production of TGF-beta and down regulation of MHC-I and II expression, similar to TVT. However, unlike TVT, spontaneous regression has not been reported in DFTD, and mortality occurs in all affected animals via starvation and frequent metastasis.8 Other transmissible tumors include clam leukemia of soft shell clams and the contagious reticulum cell Preputial mass, dog. Nuclei are mildly anisokaryotic with sarcoma of Syrian hamsters first described prominent nucleoli. Few lymphocytes are present in 6,8,11 between neoplastic cells. (Wright-Giemsa, 600X) in the 1960’s.

3 inoculation. As mentioned by the Contributing Institution: contributor, metastasis uncommonly occurs Arizona Veterinary Diagnostic Laboratory in the draining lymph node.9 University of Arizona 2831 N. Freeway After transmission, tumorous lesions Tucson, AZ 85705 typically appear within two months. http://azvdl.arizona.edu/ Conference participants discussed the pathogenesis of the tumor growth, stabilization, References: and regression phases. The initial growth stage is called the progressive phase (P). 1. Ganguly B, Das U, Das AK. Canine During the P phase, almost all TVT cells transmissible venereal tumor: A lack expression of major histocompatibility review. Comp Oncol. 2016; 14(1):1- complex (MHC) I and II due to production 12. of inhibitory cytokine transforming growth 2. Gross TL, Ihrke PJ, Walder EJ, factor-beta (TGF-beta). This allows the TVT Affolter VK. Skin Diseases of the cells to grow and evade immune destruction Dog and Cat. 2nd ed. Oxford, UK: by cytotoxic T-lymphocytes. After three to Blackwell Publishing; 2005:800-803. nine months, the tumor stabilizes and begins 3. Komnenou AT, Thomas AL, to spontaneously regress. During the Kyriazis AP, et al. Ocular regression (R) phase, interleukin-6 (IL-6) manifestations of canine trans- from infiltrating lymphocytes is thought to missible venereal tumor: A work in conjunction with interferon-gamma retrospective study of 25 cases in (IFN-gamma) to antagonize TGF-beta and Greece. Vet Rec. 2015; 176(20):523- increase MHC I and II expression on TVT 527. cells.1,8-10 4. Marcos R, Santos M, Marrinhas C, et al. Cutaneous transmissible venereal In addition to TVT, conference participants tumor without genital involvement in also discussed the devil facial tumor disease a prepubertal female dog. Vet Clin (DFTD), which is an emerging rapidly fatal Pathol. 2006; 35:106–109. transmissible tumor that is decimating the 5. Murchison EP, Wedge DC, et al. wild Tasmanian devil population.8,11 This Transmissible dog cancer genome tumor is of Schwann cell origin and is reveals the origin and history of an

ancient cell lineage. Science. 2014; polymerase chain reaction for canine 343:437-440. transmissible venereal tumor 6. Ostrander EA, Davis BW, Ostrander (CTVT) diagnosis. J Vet Med Sci. GK. Transmissible tumors: Breaking 2016; 78(7):1167-1173. the cancer paradigm. Trends Genet. 11. Siddle HV, Kaufman J. A tale of two 2016; 32:1-15. tumours: Comparison of the immune 7. Park MS, Kim Y, Kang MS, et al., escape strategies of contagious Disseminated transmissible venereal cancers. Mol Immunol. 2013; tumor in a dog. J Vet Diagn Invest. 55:190-193. 2006; 18(1):130-133. 12. VonHoldt BM, Ostrander EA. The 8. Pye RJ, Woods GM, Kreiss A. Devil singular history of a canine facial tumor disease. Vet Pathol. transmissible tumor. Cell. 2006; 2016; 53(4):726-736. 126(3):445-447. 9. Schlafer DH, Foster RA. Female genital system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s

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2016:448-449.

10. Setthawongsin C, Techangamsuwan S, Tangkawattana S, et al. Cell-based

Case 2 - Clinicopathologic Data

Case 3 - Clinicopathologic Data

Initial blood work: BUN: 26 mg/dL [reference range: 5-20] Creatinine: 2.0 mg/dL [reference range: 0.6-1.6] Blood glucose: WNL ALT (458 IU/L [reference range: 10-55]).

Urinalysis: Glucosuria (4+) Her urine was isosthenuric and contained fine granular casts.

Subsequent blood work one month later: BUN: 23 mg/dL; Serum creatinine: 3.0 mg/dL ALT (707 IU/L)

Urinalysis: Mild proteinuria (UPC: 1.3 [normal <0.5]) SDS-PAGE analysis demonstrated proteinuria due to the presence of low molecular weight protein. An aliquiot from the urine sample documented generalized amino aciduria and glucosuria without ketonuria.