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Joint Pathology CenterVeterinary Pathology Services

WEDNESDAY SLIDE CONFERENCE 2016-2017

C o n f e r e n c e 1 24 August 2016

CASE I: 48422 (JPC 4006379).

Signalment: 4-year-old male castrated sableferret (Mustela putorius furo).

History: The ferret presented with a one-week history of ataxia. The owner noted amass on the left neck. The ferret was eatingbut otherwise lethargic. Owners electedeuthanasia due to worsening of clinical signsand the inoperable nature of the mass.

Gross Pathology: Within the subcutis andskeletal muscle of the neck just caudal to theskull, there is a large, firm, multinodular,smooth, white to tan, 5 x 3.5 x 1.5 cm massextending to the left lateral and ventralaspect of the neck. The mass is adhered tothe first cervical vertebra, extendingcaudally to the mid-cervical region andventrally to the level of the trachea, but isnot adhered to the trachea or esophagus. Thecentral region of the mass is attenuated.After removal of the skull, evaluation of C1reveals that the mass infiltrates into andeffaces the left cranial articular fovea(articular facet of the atlas). Afterdecalcification, further sectioning of themass reveals that it extends into thevertebral canal and compresses the cervicalspinal cord.

In the caudal aspect of the left cranial lunglobe, a 0.5 cm diameter firm, smooth, whitenodule is observed. The spleen is diffuselycongested and enlarged, and contained a 2 x2 x 1.2 cm soft, smooth, red nodule. Tworenal cortical cysts are observed in the rightkidney, measuring approximately 0.4 cmeach.

Laboratory results: N/A

Histopathologic Description: A de-calcified transverse section through thecervical vertebra, cervical spinal cord and

Cervical vertebrae, ferret. A large mass is present overthe cranial cervical vertebrae. (Photo courtesy of: AnimalMedical Center, 510 East 62nd St. New York, NY 10065http://www.amcny.org/)

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surrounding skeletal muscle is examined.Infiltrating into the epaxial musculature,cervical vertebral pedicle, articular pro-cesses and extending into the vertebral canalwith compression of the adjacent cervicalspinal cord is a multilobular, infiltrative,poorly demarcated neoplasm. Neoplasticlobules are separated by eosinophilic fibro-vascular connective tissue. Lobules arecomprised of large polygonal cells withdistinct cell borders and abundant, am-phophilic to clear, vacuolated cytoplasm(physaliferous cells) surrounded by variableamounts of amphophilic, mucinous stroma.Nuclei are round to oval and often per-ipheralized, with finely stippled chromatinand multiple nucleoli. There are zero mitoticfigures in 10 high power (400x) fields.Multifocally in the center and at theperiphery of lobules, scattered aggregates ofcartilage and woven bone are interspersedwithin the physaliferous cells. Bony tra-beculae contain osteocytes and are lined byosteoblasts. Chondrocytes lie within alightly eosinophilic to amphophilic matrix orare occasionally entrapped within bone.Rare binucleate cells are found. Smallaggregates of lymphocytes, plasma cells andmacrophages are present at the periphery of

the mass.

Vertebral bone has scalloped borders, withosteoclasts in Howship’s lacunae. Frag-mented woven bone (reactive bone) isinterspersed with numerous plump ost-eoblasts and fibroblasts. Neoplastic com-pression of the cervical spinal cord results inshifting of the ventral medial fissure awayfrom the mass. The white matter of thespinal cord exhibits mild, multifocal vac-uolation in the dorsal, lateral and ventralfuniculi, within which are few Gitter cellscontaining myelin degradation products.Scattered, multifocal swollen axons (sph-erocytes) are present in the ventral andlateral funiculi. Variation in neuronalcytoplasmic staining is present with no lossof Nissl substance (decalcification artifact,presumptive). Surrounding epaxial skeletalmuscles, and more prominently, muscleadjacent to the dorsal spinous process un-dergoes degeneration and regeneration,manifesting as myocyte size variation, sar-coplasmic hypereosinophila and vac-uolation, loss of striation, satellitosis, centralnuclei, and nuclear rowing.

Contributor’s Morphologic Diagnoses: 1.Cervical spine: Chordoma, with local

Cervical vertebra, ferret. This fixed specimen (likelyproximal C-3 with dorsal spinous process at 10 o’clock) isunilaterally effaced by a multilobular mass which alsocompresses the spinal cord. (Photo courtesy of: AnimalMedical Center, 510 East 62nd St. New York, NY 10065http://www.amcny.org/)

Cervical vertebra (presumptive axis), ferret. The cervicalvertebra is partially effaced by the chordoma, whichcompresses the spinal cord. (HE, 5X)

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infiltration of skeletal muscle, vertebral boneand vertebral canal.2. Cervical spinal cord: Focal compressionwith midline shift, mild, moderate Walleriandegeneration and spheroid formation.3. Cervical skeletal muscles: Multifocalmyodegeneration and regeneration.4. Lung (caudal aspect left cranial lung lobe,not submitted): Multifocal chordomametastasis.

Contributor’s Comment: Chordomas arethe 5th most common tumor in domesticferrets, and the most common mus-culoskeletal tumor.1,11 Chordomas are slowgrowing, locally aggressive neoplasms der-ived from remnant fetal notochord 1,4,6-12,14

believed to originate from the primitivemesoderm.1,2 The notochord persists be-tween the vertebrae and expands to form thenucleus pulposus of intervertebral disks insome animals (visible in the section).1,5,8,9

Cell rests of residual notochord remainoutside of the intervertebral disks in anestimated 2% of humans, and chordomas arethought to arise from these cell rests.1,4,8,9

Histologically, ferret chordomas are cha-racterized by lobules of physaliferous cellssurrounding cartilage with a central core ofbone and cartilage. 1,4,6-12,14 Notochord-induced differentiation of bone and cartilagewithin the neoplasm is one hypothesis forthe bone and cartilage formation withinthese neoplasms.4

Chordomas can occur anywhere along theaxial skeleton. In the ferret, common sitesinclude the tail tip (most common site),cervical vertebral column (second mostcommon site), thoracic vertebral column,and the coccygeal region.1,4,6-12,14 The sacro-coccygeal and spheno-occipital regions arethe most common sites of chordomaoccurrence in humans, with the remainder

Cervical vertebra (presumptive axis), ferret. The neoplasm is composed of polygonal cells with large clear vacuoles whichoften peripheralize the nucleus (physaliferous cells) separated by moderate amounts of blue mucinous matrix (arrow). (HE,400X)

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found in the vertebral axis.4,14 Up to 30% ofhuman chordomas are reported to met-astasize, typically during the phase ofrecurrent disease, with common sites ofmetastasis reported to be bone, lung, lymphnode, skin and liver.1,4,14 Rare cases of localand distant skin metastases have beenreported in the ferret.1,4,14 Vascular invasionwas equivocal in the submitted section,however, was observed rarely in othersections of the mass and pulmonary met-astases were present. One study evaluatedcell proliferation indices in 5 ferretchordomas, and found that they were notpredictive of metastatic potential.8

In humans, there are 3 recognized subtypesof chordoma: classic chordoma, chondroidchordoma, and chordoma with malignantspindle cell component (dedifferentiated). Ithas been suggested that ferret chordomasmay act as an animal model for thechondroid subtype of human chordoma.4,14

Differentiation between classic and cho-ndroid subtypes in humans is significant, aschondroid chordomas are associated with asurvival rate 3 times greater than that ofclassic chordomas.1,8 Although the cho-ndroid component was not prominent in thesubmitted section, it was more frequentlyobserved in other sections of the mass.

Immunohistochemical analysis of cho-rdomas in the ferret typically show dualexpression of cytokeratin and vimentin, withvariable expression of S-100 protein andneuron specific enolase (NSE).1,4,6-12,14 Inone study of chordomas in 20 ferrets, 75%were positive for S-100 and 85% werepositive for NSE.4 The S-100 positivity isthought to be due to glycosaminoglycancontent within the stroma, and NSE isexpressed in cells with high metabolicactivity.1,4

JPC Diagnosis: Cervical vertebra (axis):Chordoma.

Conference Comment: This case nicelycharacterizes an example of a commonneoplasm in the axial skeleton of a ferret.The contributor provides an excellent his-tologic description, including the ancillaryfindings of degeneration of the spinal cord,peripheral nerves, and adjacent skeletalmuscle as a result of compression from thisinfiltrative neoplasm.

Aside from humans and ferrets, chordomashave been rarely reported in the axialskeleton of Fischer 344 rats11, mice13,dogs6,9, and cats2. In addition, 24 cases ofspontaneous primary intestinal chordomas aswell as nine spontaneous vertebral chor-domas, were recently reported in the agedlaboratory zebrafish3. In dogs, chordomashave been reported in the brain, spinal cord,and skin6. Of the three reported cases offeline chordoma, one was initially diagnosedas chronic granulomatous inflammation dueto the interpretation of the characteristicphysaliferous cells as atypical, foamy mac-rophages; it was subsequently dis-covered tobe an example of a classic chordoma-likesubtype, which then metastasized to multiplelymph nodes2.

Cervical vertebra (presumptive axis), ferret. Multifocally,several lobules of the neoplasm contain foci ofmineralizing woven bone. (HE, 200X)

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In all species, chordomas tend to be slowgrowing and locally invasive.1-14 Typically,distant metastasis is associated with theclassic chordoma and dedifferentiatedspindle cell subtypes in humans, rather thanthe chondroid type which forms islands ofcartilage and bone most commonly in theferret and mink.1,4,8,10,14 In a large study ofFischer 344 rats there was an exceptionallyhigh 75% incidence of pulmonary metastasisin animals diagnosed with chordomas.11 Thehistomorphology of chordomas in theFischer rat is similar to the classic type inhumans, which may explain the moreaggressive biologic behavior in theserodents as compared to other animals.11

Prior to this case, visceral metastasis had notyet been reported in the ferret. However, thecontributor recently published this casereport, to include pulmonary metastasis viahematogenous spread; this is the first knowncase of visceral metastasis in the ferret5. Theauthors hypothesized that the metastaticpotential in ferrets may increase over time,highlighting the importance of promptsurgical excision. Most cases of ferretchordomas occur at the tail tip makingcomplete surgical excision relativelysimple.1,4,5,14 Chordomas in other locations,such as this case, are substantially moredifficult to excise.5

Chordomas in ferrets may histologicallyresemble chondrosarcomas due to thecartilaginous component. Conference par-ticipants briefly discussed imm-unohistochemistry as a method by which todifferentiate chordoma from cho-ndrosarcoma. As mentioned by the con-tributor, chordomas consistently dem-onstrate dual immunopositivity for vimentinand cytokeratin, while chon-drosarcomas areimmunonegative for cytokeratin. Chordomasare also typically positive for S-100 andneuron-specific enolase (NSE); the S-100and NSE positivity does not suggest neural

crest origin, however.10,12 Other neoplasmsof interest that typically express bothvimentin and cytokeratin include: mes-othelioma, synovial sarcoma, meningioma,renal cell carcinoma, adrenal carcinoma, andendometrial sarcoma.12

Contributing Institution: The AnimalMedical CenterDepartment of Pathology510 East 62nd St. New York, NY 10065http://www.amcny.org/

References:1. Camus MS Rech RR, Choy FS, FiorelloCV, Howerth EW. Pathology in practice,chordoma on the tip of the tail of a ferret. JAm Vet Med Assoc. 2003;235(8):949-51.2. Carpenter JL, Stein BS, King NW Jr,Dayal YD, Moore FM. Chordoma in a cat. JAm Vet Med Assoc. 1990;197:240-242.3. Cooper T, Murray K, et al. Primaryintestinal and vertebral chordomas inlaboratory zebrafish (Danio rerio). VetPathol. 2015;52(2):388-392.4. Dunn DG, Harris RK, Meis JM, SweetDE. A histomorphologic and immune-ohistochemical study of chordoma in twentyferrets (Mustela putorius furo). Vet Pathol.1991;28:467-473.5. Frohlich J, Donovan T. Cervicalchordomas in a domestic ferret (Mustelaputorius furo) with pulmonary metastasis. JVet Diagn Invest. 2015;27(5)656-659.6. Gruber A, Kneissi S, Vidoni B, UrlA. Cervical spinal chordoma withchondromatous component in an dog. VetPathol. 2008;45(5):650653.7. Koestner A, Bilzer T, Fatzer R, SchulmanFY, Summers BA, Van Winkle TJ.Chordoma. In: Histological Classification ofTumors of the Nervous System of DomesticAnimals, 2nd series, vol. V, pp. 36. ArmedForces Institute of Pathology, Washington,D.C., 1999.

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8. Munday JS Brown CA, Richey LJ.Suspected metastatic coccygeal chordoma ina ferret (Mustela putorius furo). J Vet DiagnInvest. 2004;16(5):454-458.9. Munday JS, Brown CA, WeissR. Coccygeal chordoma in a dog. J VetDiagn Invest. 2003;15(3):285-288.10. Pye GW, Bennett RA, Roberts GD,Terrell SP. Thoracic vertebral chordoma in adomestic ferret (Mustela putorius furo). JZoo Wildl Med 2000;31(1):107-111.11. Stefanski SA, Elwell MR, Mitsumori K,Yoshitomi K, Dittrich K, GilesHD. Chordomas in Fischer 344 rats. VetPathol. 1988;25(1):42-47.12. Takeshi Y, Tetsuo O, et al.Histochemical and immunohistochemicalcharacterization of chordoma in ferrets. JVet Med Sci. 2015;77(4):467-473.13. Taylor, K, Garner M, et al. Chordomasat high prevalence in the captive populationof the endangered Perdido Key Beachmouse (Peromyscus polinotus trissyllepsis).Vet Pathol. 2016;53(1):163-169.14. Williams BH, Eighmy JJ, Berbert MH,Dunn DG. Cervical chordoma in two ferrets(Mustela putorius furo). Vet Pathol.1993;30:204-206.

CASE II: 48422 (JPC 4006379).

Signalment: 16 year old, female, domesticshorthair, Felis catus.

History: The cat had decreased appetite andweight loss. It was icteric with a temperatureof 38°C. The heart rate was 220 beats/min

and the respiration rate was 92 breaths/min.Lymph nodes were unremarkable bypalpation. Ultrasound of the abdomenshowed moderate amounts of fluid, and aliver with rounded edges and a marbledparenchyma with reduced number of visiblehepatic- and portal veins. Lymph nodes inthe cranial abdomen, near the spleen andpancreas were enlarged and hypoechoic.

Gross Pathology: The cat was thin andicteric. The abdominal cavity contained 50ml clear yellow fluid with a few floccules.The liver was slightly enlarged with roundededges and lighter in color than normal with alobular pattern and a decreased texture.Multifocally scattered, light gray, softnodules, 0.5-5.0 mm in diameter werepresent in the hepatic parenchyma. Themesenteric lymph node was enlarged, lightyellow-gray, moderately firm, and measured4.0x1.5x1.5 cm. The pancreas wasmacroscopically normal.

Laboratory results: Clinical pathology: AST:193 U/L (ref: 0-60), ALT: 258 U/L (ref: 0-75),AP: 339 U/L (ref: 0-40), amylase: 880 U/L (ref:0-800), total protein: 56 g/L (ref: 60-82),albumin: 21 g/L (ref: 25-39), bile acids: 392µmol/L (ref: 0-5), total bilirubin: 215 µmol/L(ref: 0-4), WBC: 4.2 x 10e9/L (ref: 5.5-17),lymphocytes: 0.6 x 10e9/L (ref: 1.5-7.0),platelets: 30 x 10e9/L (ref: 180-400).

Immunohistochemical staining of mesentericlymph node and liver showed immunoreactivitywith rabbit anti-human CD3 (DakoCytomation,A 0452) and confirmed that the infiltrating cellswere T lymphocytes.

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Histopathologic Description: In the liver,there are multifocal to confluent, randomlyspaced areas with numerous infiltratingsmall lymphoid cells. The cells are locatedboth in the sinusoids and appear to belocated within the cytoplasm of hepatocytes.Some of the cells are also located close to orin invaginations of the hepatocellular mem-brane. The cells are small, with a narrow rimof eosinophilic cytoplasm. They have asmall, densely stained round to irregularnucleus with indistinct nucleolus. There islittle degree of anisocytosis and an-isokaryosis. There is <1 mitotic figure per10 HPF. The lymphoid cells present withinthe hepatocellular cytoplasm are surroundedby a clear halo. There is severe dissociationof hepatocytes, seen as single rounded largehepatocytes with up to 8 lymphoid cellswithin the cytoplasm. In areas with fewlymphoid cells, there are moderate vac-uolation of hepatocytes with variably sizedclear vacuoles.

Contributor’s Morphologic Diagnoses:Liver: malignant lymphoma with emp-eripolesis of lymphocytes and severedissociation of hepatocytes

Contributor’s Comment: Feline lymphomacommonly occurs as thymic, alimentarymulticentric and cutaneous syndromes.8 This

cat had involvement of the mesentericlymph node and liver. It is indicated thatFeline leukemia virus (FeLV) positive catsare associated with the mediastinal andmulticentric form of lymphoma and that thethymic and alimentary forms predominate inFeLV negative cats.2,8 The FeLV status ofthis cat was not known.

Hepatic lesions morphologically identical tothose in this cat, has been reported earlier intwo cats diagnosed with lymphoblasticlymphosarcoma.3Although emperipolesis isthe phenomenon of viable cells entering the

cytoplasm of other cells without damage toeither host cell or the engulfed cells,3 theclinical pathology of this cat, similar to thecats presented by Ossent et al,5 indicatessevere hepatic failure. In addition, histologyof the liver shows extensive hepatocellulardissociation.

JPC Diagnosis: 1. Liver: T-cell lymphoma,hepatocytotropic.2. Liver: Regeneration, micronodular,multifocal, mild.

Liver, cat. Hepatic cords are diffusely dissociated andhepatocytes contain multiple lymphocytes within theircytoplasm. Intrahepatocytic lymphocytes are surroundedby a clear vacuole. (HE, 400X)

Liver, cat. Intrahepatocytic lymphocytes stain strongly

positive with anti-CD-3 immunohistochemical stain.

(anti-CD3, 400X) (Photo courtesy of: Norwegian School

of Veterinary Science, Institute of Basic Sciences and

Aquatic Medicine, PO box 8146 Dep, 0033 Oslo Norway.

http://www.veths.no/Venstremeny/English/)

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Conference Comment: This case generateddiscussion amongst the conference par-ticipants regarding the use of the termemperipolesis. As mentioned by the con-tributor, emperipolesis is an uncommonbiological process, in which a cell penetratesthe cytoplasm of another living cell. 1,4,6,7

Unlike phagocytosis where the engulfed cellis killed by lysosomal enzymes of themacrophage, the engulfed cell exists as aviable cell within another.7 The pathogenesisand significance of this phenomenon iscurrently unknown.1 Emperipolesis has beenrarely reported in the human literatureassociated with autoimmune hemolyticanemia, multiple myeloma, leukemia, andmalignant lymphoma.1 In this case, there isclear evidence of hepatocellular injury andcholestasis supported by the animal’s cl-inical icterus along with severe serum bio-chemistry abnormalities. Clinicopathologicserum elevations in AST and ALT, as wellas hypoalbuminemia and hypoproteinemiasupport hepatocellular injury in addition tohepatocellular swelling, lipofuscin dep-osition, and hepatic lipidosis. Cholestasis is

confirmed by severe elevations in bile acids,total bilirubin, and elevated ALP.

Given the degree of hepatocellular injuryand cholestasis, conference participantsunanimously preferred the term hep-atocytotropism over emperipolesis as a morerepresentative term for the infiltrating T-celllymphocytes in this case. This terminologywas recently proposed as an alternative toemperipolesis in similar cases malignantlymphoma in the dog with distinct tropismfor hepatocytes.4 Transmission electronmicroscopy in the dog as well as thepreviously reported cases in cats, showedlymphocytes within invaginations of thehepatocyte cell membrane rather than withinthe cytoplasm, as required for em-peripolesis.4,6,7 In addition, hepatocyto-tropism is more analogous to the epi-theliotropism present in cutaneous andintestinal T-cell lymphoma in the dog andcat.4

Several conference participants alsocommented on the degree of hepatic corddiscohesion with dislocation of hepaticplates and diffuse loss of normal architecturein this case. Some speculated this to bepartially due to moderate autolysis of thetissue. There was further discussion and

Liver, cat. In areas of marked hepatic cord dissociation,Kupffer cells contain abundant granular brown pigment,which likely represents a combination of iron (confirmedwith Perl’s stain), bile pigment, and lipofuscin fromhepatocellular damage. (HE, 400X)

Liver, cat. Scattered throughout the section are rarenodules of proliferating hepatocytes which contain fewintrahepatocytic lymphocytes (arrows). The mild lipidosisseen diffusely through the section is best demonstratedhere. (HE, 400X)

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some speculation that hepatic discohesioncould be due to a defect in β-catenin through the Wnt signaling pathway, common inhepatic neoplasms.9 This pathway has amajor role in cell adhesion to the basementmembrane and cell polarity in the liver andgastrointestinal tract. In addition, β-catenin binding to E-cadherin is responsible formaintaining intercellular adhesion.5 Nor-mally, after acute hepatocellular insult, theliver is stimulated to regenerate via the Wntpathway. The Wnt receptor signals throughcell surface receptor, frizzled (FRZ), todeactivate the destruction complex, APC.The tumor suppressor APC normally bindsto and destroys β-catenin.5 With the APCdeactivated, β-catenin signaling drives the expression of target genes that are criticalfor cell cycle progression and contribute toinitiation of the regeneration process.5,9

Even in the multifocal microregenerativenodules without hepatocytotropic lym-phocytes, there is hepatic cord discohesion.Regardless of the pathogenesis, the dis-association of the hepatic cords likelydisrupts bile canalicular transport and sec-retion of bilirubin leading to severe hepaticcholestasis and icterus in this case.6

Contributing Institution: NorwegianSchool of Veterinary ScienceInstitute of Basic Sciences and AquaticMedicineOslo, Norwayhttp://www.veths.no/Venstremeny/English/

References:1. Amita K, Shankar S, et al. Emeripolesis ina case of adult T cell lymphoblasticlymphoma (mediastinal type)-detected atFNAC and imprint cytology. Online JHealth Allied Scs. 2011;10:11.2. Fry, M, McGavin M. Bone marrow, bloodcells, and the lymphatic system. In: ZacharyJF, McGavin MD, eds. Pathologic Basis of

Veterinary Disease. 5th ed. St. Louis, MO:Elsevier; 2012:698-705.3. Humble JG, Jayne WHW, Pulvertaft RJV:Biological interaction between lymphocytesand other cells. Br J Heamatol 1956;2:283-294.4. Keller S, Vernau W, et al. Hepatosplenicand hepatocytotropic T-cell lymphoma: twodistinct types of lymphoma in dogs. VetPathol. 2012;50:281-290.5. Kumar V, Abbas A, Aster J. Neoplasia.In: Kumar V, Abbas A, Aster J, eds.Robbins and Cotran Pathologic Basis ofDisease. 9th ed. Philadelphia, PA; SaundersElsevier; 2015: 296-297.6. Ossent P, Stöckli RM, Pospischil A:Emperipolesis of lymphoid cells in felinehepatocytes. Vet Pathol. 1989;26:279-280.7. Suzuki M, Kanae Y, et al. Emperipolesis-like invasion of neoplastic lymphocytes intohepatocytes in feline T-cell lymphoma. JComp Path. 2011;144:312-316.8. Valli V, Kiupel M, Bienzle D.Hematopoietic system. In: Jubb, Kennedy,and Palmer’s Pathology of DomesticAnimals, ed. Maxie MG, 4th ed., Vol. 3.Saunders Elsevier, Philadelphia, PA,2016:103-104.9. Wang E, Yeh S. et al. Depletion of β-catenin from mature hepatocytes of micepromotes expansion of hepatic progenitorcells and tumor development. PNAS.2011;108:18384-18389.

CASE III: M (JPC 4035590).

Signalment: 16-week-old female and malemink kits, Mustela vison

History: Kits were vaccinatedsubcutaneously at 6 weeks of age with 3portions of the 4-way combination vaccineincluding inactivated mink enteritis virus,Clostridium botulinum Type C bacterin-toxoid and inactivated Pseudomonas

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aeruginosa bacterin. Kits were vaccinated atapproximately 10-12 weeks of age with themodified- live canine distemper virusportion of the 4-way combination vaccinevia aerosol spray.

Approximately 10 days later, mortalitystarted with 5- 10 dead kits per day.Mortality was concentrated in 2 of 8 barnsand the mahogany mink were primarilyaffected. Affected mink kits had “wetheads” and were screaming, seizuring beforedying. This producer mixes his own feed onfarm and in the last week, had a feed changeto fish, chicken and processed meats. Onlythe chicken is fresh daily, the other proteinsources are frozen. As of a week ago,antibiotics (CSP 250) were mixed in thefeed. The older mink are fine.

Gross Pathology: Four mink kits weresubmitted for postmortem examination.They were in good body condition withample internal and external fat stores. Theywere mildly to moderately dehydrated. Thelungs were diffusely mottled purple/red andwere edematous. The tracheal lumina didnot contain fluid or exudate. Stomachs weregenerally either empty or contained brownflecked mucus or brown fluid. The smallintestines were generally empty and only a

small amount of gold brown mucoid to pastymaterial was in the rectum. Livers were redtinged with yellow. The spleens were small.

Laboratory results: Negative for Aleutiandisease by CIE testing. Multiple tissuesincluding lung, spleen, liver, adrenal gland andbrain had positive immunohistochemical (IHC)staining for canine distemper virus (CDV).

Histopathologic Description: The lung ismarkedly congested with alveoli containingeosinophilic edema fluid, small amounts offibrin and red blood cells. Multifocally,small to moderate numbers of macrophageswith abundant often foamy cytoplasm, lessfrequently multinucleated cells and in-frequent neutrophils are also collecting inalveolar spaces. Bronchial lumina containeosinophilic edema fluid, small numbers ofred blood cells, macrophages, multinucleatecells, neutrophils and degenerate cells.Many of the bronchi and nearby vessels arerimmed by one to two cell thick lym-phohistiocytic cuffs. Numerous largeeosinophilic spherical to rectangular in-clusions morphologically compatible withviral inclusions are present within thecytoplasm of normal and proliferative bro-nchial and bronchiolar epithelium andoccasionally within macrophages or mul-tinucleated cells. Strong positive staining forCDV is present within bronchiolar ep-ithelium and alveolar macrophages onimmunohistochemistry.

Contributor’s Morphologic Diagnoses: 1.Marked pulmonary congestion and moderatepulmonary edema.2. Proliferative bronchitis, bronchiolitis andmultifocal histiocytic pneumonia witheosinophilic intracytoplasmic inclusions.3. Very mild acute suppurative bronchitis,bronchiolitis and pneumonia

Contributor’s Comment: The host range ofcanine morbillivirus (canine distemper virus,

Lung, mink. At subgross magnification, the section oflung is markedly congested. (HE, 5X)

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CDV) is broad and species in all families inthe order Carnivora, including Canidae,Mustelidae, Procyonidae, Hyaenidae,Ursidae , Viverridae and Felidae areconsidered susceptible.9 Canine distempervirus causes multisystemic disease in farmedmink4 with juvenile mink being mostsusceptible with a mortality rate of 90% ascompared to a more variable rate of 20-90%

in adult mink.9 Outbreaks of CDV infectionon commercial mink farms have beenassociated with infected raccoons (Procyonlotor) and striped skunks (Mephitismephitis) found on the farm premises4. Thetransmission of the virus is mainly byaerosol or through direct contact with oral,nasal or conjunctival fluid as the caninedistemper virus does not survive long in theenvironment and it can be rapidly in-activated by ultraviolet light, drying, heatand common disinfectants. However, it ispossible for the gloves used for handlinginfected mink to act as fomites and transmitsufficient virus to infect susceptible mink fora short period of time.2

Routine vaccination of kits via subcutaneousinjection using a multivalent vaccineincluding canine distemper virus is typicallycarried out after weaning.4 Some producerswill also revaccinate the mature females andmales being retained for breeding at thistime (J Mitchell, personal communication).On this farm, the 4-way combinationvaccine was split and the mink kits were

immunized by subcutaneous injection formink enteritis virus, Clostridium botulinumType C and Pseudomonas aeruginosa at 6weeks of age. Four to six weeks later, thekits were given the canine distemper virusportion of the vaccine by aerosol spray,however, as the vaccine is meant to be givenby subcutaneous injection it is likely themink kits were not sufficiently immunizedagainst canine distemper virus andsubsequently developed clinical disease.

At necropsy, the most consistent lesionswere identified in the lungs with pro-liferative bronchitis and bronchiolitis andvariable numbers of large rectangular

Lung, mink. There is marked hyperplasia of bronchiolar epithelium which extends into the surrounding alveoli. Viralsyncytia may be seen in lumen of the affected airway as well as within macrophages in surrounding alveoli. (HE, 5X)

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eosinophilic intracytoplasmic inclusionsidentified in bronchial and bronchiolarepithelium. Low numbers of multinucleatedcells in alveoli occasionally also contain in-tracytoplasmic inclusions. Small accum-ulations of neutrophils were aggregating insmall airways and secondary bacterial bron-chopneumonia is reported to commonlyoccur.4 The diagnosis of CDV in theseindividuals was confirmed with IHC.

In experimental infections with the virusgiven by injection, young mink are reportedto develop reddening of the skin progressingto dermatitis, conjunctivitis and nasal dis-charge, but dyspnea is uncommon.4 How-ever, histologically, pulmonary lesions arecommon and the intracytoplasmic andintranuclear viral inclusions are most oftenpresent in the respiratory or bladder ep-ithelium.4 Viral inclusions are not identifiedin the transitional epithelium of the bladderin this case, despite the tremendous numbersvisible in the lung tissues.

JPC Diagnosis: Lung: Bronchiolarepithelial hyperplasia, marked, with mildhistiocytic bronchiolitis, and epithelial andhistiocytic intracytoplasmic viral inclusionbodies.

Conference Comment: Canine distempervirus (CDV) is an enveloped, singlestranded RNA (ssRNA) virus of the genusMorbillivirus in the Paramyxoviridaefamily1-8. Morbilliviruses are highly con-tagious pathogens that are responsible forsome of the most devastating diseases thataffect mammals worldwide. Other membersof this genus include measles virus, pestedes petits ruminants virus, rinderpest virus,phocine distemper virus, and dolphin

morbillivirus.6 CDV is one of the mostimportant and ubiquitous infectious diseasesof wild and domestic predators with highmorbidity and mortality in imm-unologicallynaïve populations.5 As men-tioned by thecontributor, mustelids (i.e. ferrets, minks,and badgers) are particularly sensitive to thevirus with mortality up to 100% in non-vaccinated ferrets.5

Like all paramyxoviruses, CDV contains sixstructural proteins: nucleocapsid (N),phospho (P), large (L), matrix (M), hem-agglutinin (H), and fusion (F) protein. Theviral envelope contains H, used forattachment to the host cell receptor, and F,required for entry and syncytia formation. InLung, mink. Bronchiolar epithelium contains one or

multiple 2-4um irregularly round intracytoplasmic viralinclusions. (HE, 320X)

Lung, mink. Alveoli contain numerous macrophageswhich occasionally contain intracytoplasmic viralinclusions (arrow). A multinucleated viral syncytium ispresent at right. (HE, 400X)

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this case, syncytial cells are occasionallypresent within bronchioles and alveoli.1,6

CDV is typically transmitted via inhalationof infected aerosols.3,4 The virus entersmacrophages, lymphocytes, and dendriticcells via the signaling lymphocyte activationmolecule (SLAM) receptor binding to Hviral protein within the first day ofinfection.1,6 The virus spreads to local lymphnodes and other lymphoid organs within twoto five days post infection. Primary viralreplication in the lymphoid tissues leads tosevere immunosuppression and viremia.About eight to ten days post-infection, CDVdisseminates to several epithelial tissues(respiratory, intestinal, and urinary) and thecentral nervous system. The virus usesepithelial cell receptor nectin-4 to gain entryinto epithelial cells.1,4,6

Unfortunately, in this case, the conferenceparticipants had difficulty assessing thealveolar septal changes due to poorinsufflation of the lung. However, con-ference participants easily identified

numerous intracytoplasmic viral inclusionswithin histiocytes and the markedlyhyperplastic bronchiolar epithelium. Inaddition, some recognized rare intranuclearinclusions. CDV is unique because it causesboth intranuclear and intracytoplasmicinclusions.3,7,8 Given that CDV is an ssRNAvirus that requires RNA-dependent RNApolymerase complex present only in thecytosol to replicate, the presence of int-ranuclear inclusions is initially con-founding.7 However, CDV infection inducesthe cellular stress response which causeselevated expression of heat shock proteins.These proteins translocate viral nucleocapsid(N) proteins from the cytoplasm into thenucleus as part of the normal host cellresponse to cellular stress. Under normalcircumstances, the N protein would be toolarge to pass through nuclear pores.7 Nproteins form the basis for nuclear bodyformation, and propagation of viral nuc-leocapsids. These particles eventuallypartially, or completely fill the nucleusresulting the in the formation of theintranuclear inclusion body visible by lightmicroscopy.7,8

The contributor mentions the wide range ofspecies now infected by CDV and relatedviruses. Additionally, lethal outbreaks inrhesus and cynomolgus macaques ori-ginating in China have been documentedwithin the last decade.

Contributing Institution: Animal HealthLaboratoryUniversity of Guelph, Guelph, Ontario,Canadahttp://ahl.uoguelph.ca

References:1. Beineke A, Baumgartner W, Wohlsein P.Cross species transmission of canine

Lung, mink. In this field, numerous alveolarmacrophages contain variably-sized viral inclusions(arrows). (HE, 400X)

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distemper virus-an update. One Health.2015; 1:49-59.2. Budd J. Distemper. In: Infectious diseasesof wild mammals. 1st ed. Ames, IA: IowaState Press; 1970:36-49.3. Caswell J, Williams K. Respiratorysystem. In: Maxie MG, ed. Jubb, Kennedy,and Palmer’s Pathology of DomesticAnimals. Vol 2. 6th ed. Philadelphia, PA:Elsevier Saunders; 2016: 574-575.4. Hunter DB. Respiratory System of Mink.In: Hunter B, Lemieux N, eds. Mink:Biology, Health and Disease. Guelph,Ontario: Graphic and Print Services,University of Guelph; 1996:13-1 – 13-15.5. Kuipel M, Perpinan D. Viral Diseases inFerrets. In: Biology and Diseases of theFerret. 3rd ed. Ames, IA: Wiley Blackwell;2014:439-450.6. Noyce R, Depeut S, Richardson C. Dognectin-4 is an epithelial cell recepotor forcanine distemper virus that facilitates virusentry and syncytia formation. Virology.2013;436:210-220.7. Oglesbee M. Intranuclear inclusions inparamyxovirus-induced encephalitis: ev-idence for altered nuclear body differen-tiation. Acta Neuropathol. 1992;84:407-415.8. Oglesbee M, Krakowa S. Cellular stressresponse induces selective intranuclear traf-ficking and accumulation of morbillivirusmajor core protein. Lab Invest. 1993;68:109-107.9. Williams ES. Canine Distemper.In: Williams ES, Barker IK, eds. Infectiousdiseases of wild mammals. 3rd ed. Ames, IA:Iowa State Press; 2001:50-59.10. Qiu W1, Zheng Y, Zhang S, Fan Q, LiuH, Zhang F, Wang W, Liao G, Hu R. Canine

distemper outbreak in rhesus monkeys, China.

Emerg Infect Dis. 2011 Aug;17(8):1541-3.11. Sakai K1, Nagata N, Ami Y, Seki F, SuzakiY, Lethal canine distemper virus outbreak incynomolgus monkeys in Japan in 2008. J Virol.2013 Jan;87(2):1105-14.

CASE IV: 09-1045 (JPC 3164901).

Signalment: 5 year old, ovariohyster-ectomized female Cavalier King Charlesspaniel, Canis familiaris.

History: An adult spayed female CavalierKing Charles Spaniel reported to be 5 yearsold from a the household with multiple dogsexhibiting increased respiratory rate andcough. During thoracic radiographs thepatient became agonal and arrested despiteattempted cardiopulmonary resuscitation.Radiographs revealed pneumonia. The pat-ient was presented for necropsy 1.5 hoursfollowing death

Gross Pathology: The trachea is diffuselymildly compressed dorsoventrally. Ex-cluding a limited regional portion of thecranial aspect of the left cranial lung lobe,the lungs are diffusely dark red, heavy, andsink in formalin. The heart is mildly

enlarged with mild nodular thickening of themitral valve. A small portion of the cer-ebellar vermis protrudes through a mildlynarrowed and irregular foramen magnum.

Lung: At subgross magnification, the lung is markedlycongested, diffusely hypercellular, and there are bloodvessels are surrounded by perivascular cuffs. (Photocourtesy of: University of Tennessee College ofVeterinary Medicine, Department of Pathobiology, 2407River Drive, Room A201, Knoxville, TN 37996http://www.vet.utk.edu/)

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Laboratory results:

MicrobiologySample: LungTest: Aerobic CultureResult: No growth seen in 3 days

PCR/DNA sequencing99 % nucleotide identity with Lycoperdonpyriforme (AY854075.1, strain AFTOL-ID-48) over 682 bp.

Histopathologic Description: Lung:Alveoli and bronchioles frequently are filledwith macrophages, neutrophils, cellulardebris, erythrocytes, multinucleate giantcells and edema. The macrophages arefrequently vesciulate, occasionally

containing cellular and karyorrhectic debris.Random, individual to multiple, golden-brown, 3-5μm diameter fungal spores are present both within macrophages and freewithin the alveolar lumens. Alveolar septaare occasionally lined by finely fibrillar to

glassy eosinophilic material (hyalin mem-branes). Alveolar epithelial cells aremultifocally cuboidal (type II pneumocytehyperplasia) with rare binucleate andmultinucleate cells. There is multifocal lossof the bronchiolar epithelium, with in-frequent epithelial dysplasia. A few peri-bronchiolar macrophages contain blackgranular pigment (anthracosis). Moderatenumbers of plasma cells with fewer lymp-hocytes surround pulmonary vessels.Vascular endothelial cells are frequentlyhypertrophic. Pleural mesothelial cells arehypertrophic.

Contributor’s Morphologic Diagnosis:Marked, diffuse, histiocytic interstitialpneumonia with fungal spores.

Contributor’s Comment: The intra-histiocytic and free fungal spores within thelung were identified as spores of “puffball”mushrooms by PCR and DNA sequencing.Eleven species of puffball mushrooms

Lung, dog. Alveoli and distal airways are often lined by thick mats of fibrin (hyaline membranes). Alveoli contain moderatenumbers of neutrophils and macrophages as well as fibrin. Septa are expanded by edema, fibrin, as well as increasednumbers of circulating neutrophils. (HE, 260X)

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(Lycoperdon sp.) are indigenous to the areafrom which this case originated (greaterSmoky Mountains). Inhalation of spores hasbeen reported infrequently in associationwith pneumonia (pulmonary lyco-perdonosis) in dogs1,8 and humans7,9.

JPC Diagnosis: Lung: Pneumonia,interstitial, necrotizing, fibrinosuppurative,and histiocytic, diffuse, chronic, severe withhyaline membrane formation and rare intra-histiocytic fungal spores.

Conference Comment: Pulmonary lesionscaused by the inhalation of Lycoperdon sp.spores are theorized to result from the hosthypersensitivity response to fungal sporesacting as foreign bodies rather than aninfectious process.2,7 The failure to cultureLycoperdon from a reported case andinability of this saprophytic fungus togerminate under normal physiologic bodytemperatures supports this theory.7 Hyp-ersensitivity pneumonitis (HP), also knownas extrinsic allergic alveolitis, is caused by

intense and often prolonged exposure toinhaled organic antigens such as fungalspores, but can also include bacterialproducts and animal proteins. This disease is

commonly diagnosed in dairy cows andhorses housed indoors and usually affectmultiple animals within a group. It resultsfrom chronic inhalation of spores ofthermophilic actinomycetes (Saccharo-physpora rectivirgula) found in moldyhay.5,6 This is followed by an antibodyresponse to inhaled antigen and localdeposition of antigen-antibody complexes(Arthus reaction) as well as the formation ofmultifocal granulomas suggesting a T-cell-mediated response. This type of reactionsuggests both type III and type IVhypersensitivity response 2,5,7 (See WSC

2012-2013, conference 22, case 4 for areview of hypersensitivity reactions).

Characteristic lesions in animal and humanHP include proliferation of type IIpneumocytes and fibrosis of alveolar septaand peribronchiolar tissue. In severe cases,hyaline membranes composed of fibrin,serum proteins, and cell debris line thealveolar septa.5 This hyaline membrane

Lung, dog. Vessels throughout the sections are cuffed y 3-5 layers of lymphocytes and fewer plasma cells. (HE,280X)

Lung: Rare macrophages contain single or multiple 4-

6um golden-brown fungal spores. (HE, 400X)(Photo

courtesy of: University of Tennessee College of

Veterinary Medicine, Department of Pathobiology, 2407

River Drive, Room A201, Knoxville, TN 37996

http://www.vet.utk.edu/)

17

causes alveolar occlusion resulting in severehypoxia and death.

Conference participants noted the severenecrotizing and inflammatory changes to thelung interstitium, but had difficult timefinding the rare fungal spores present in thetissue. Interestingly, clinical disease oflycoperdonosis in humans and dogs onlyoccurs following a massive inhalation doseof puffball conidia7; however in this sectionthere is a paucity of spores present. As aresult, there was discussion of differentials

for hyaline membrane formation, which isthe most striking histologic feature in thiscase. Acute respiratory distress syndrome(ARDS) in the dog was discussed as havinga similar histopathologic appearance to thiscase. In addition to hyaline membranes, typeII pneumocyte hyperplasia, and bronchiolarepithelial necrosis is a consistent feature ofARDS. Inciting causes include: trauma,shock, disseminated intravascular coag-ulation, septicemia, smoke inhalation,

oxygen toxicity, viral infection, andstrangulation among others.4

This case was additionally studied inconsultation with the Department ofPulmonary and Mediastinal Pathology at theJoint Pathology Center, who disagreed withthe purported mechanisms discussed above,based on the morphologic changes noted inthis section. Their interpretation is that thissection of lung is diagnostic for diffusealveolar damage (DAD) due to a toxicreaction to Lycoperdon spores rather thanHP, published in a case report from thisanimal.2 DAD is the most commonlyidentified form of interstitial lung diseaseand is the histologic correlate to the clinicalcondition of ARDS, discussed by conferenceparticipants.4 The pulmonary pathologistsdescribe an intra-alveolar inflammatoryinfiltrate composed predominantly ofneutrophils and macrophages. This isadmixed with focal plasma cells andlymphocytes, hyaline membranes, and acuteinflammation of bronchiolar epithelium,along with multifocal fungal spores. Theysee no histologic evidence to support thediagnosis of HP. They also note theimportance of recognizing this entity asDAD, and not HP, due to the high mortalityrate for DAD, even with intensivesupportive treatment.

Contributing Institution: University ofTennesseeCollege of Veterinary MedicineDepartment of Pathobiologywww.vet.utk.edu/departments/path

References:1. Aleghat T, Kellett-Gregory L, VanWinkle T: Lycoperdonosis in a dog, VetPathol. 2009;46:1046.2. Alenghat T, Pillitteri C et al.Lycoperdonosis in two dogs. J Vet DiagnInvest. 2010;22:1002-1005.

Lung: Fungal spores are argyrophilic on a Gomori

methenamine silver stain. (GMS, 400X) (Photo courtesy

of: University of Tennessee College of Veterinary

Medicine, Department of Pathobiology, 2407 River Drive,

Room A201, Knoxville, TN 37996

http://www.vet.utk.edu/)

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3. Buckeridge D, Torrance A, Daly M.Puffball mushroom toxicosis (lycoperd-onosis) in a two-year-old dachshund. VetRec. 2011;168:304.4. Caswell J, Williams K. Respiratorysystem. In: Maxie MG, ed. Jubb, Kennedy,and Palmer’s Pathology of DomesticAnimals. Vol 2. 6th ed. Philadelphia, PA:Elsevier Saunders; 2016: 514.5. Husain AN. The Lung. In: Kumar V,Abbas AK, Aster JC, eds. Pathologic Basisof Disease. 9th ed. Philadelphia, PA:Elsevier Saunders; 2015:694-695.6. Lopez A. Respiratory system, media-stinum, and pleurae. In: Zachary JF,McGavin MD, eds. Pathologic Basis ofVeterinary Disease. 5th ed. St. Louis, MO:Elsevier; 2012:513.7. Munson E, Panko D, Fink, J.Lycoperdonosis: Report of Two Cases andDiscussion of the Disease: Clin MicrobiolNewsl. 1997;19(3):17-20.8. Rubensohn M: Inhalation pneumonitis ina dog from spores of puffball mushrooms.Can Vet Journal. 2009;50(1):93.9. Taft T, Cardillo R et al. Respiratoryillness associated with inhalation ofmushroom spores-Wisconsin, 1994. MMWRMorb Mortal Wkly Rep. 1994;43(29):525-526.