AFIP Wednesday Slide Conference - No. 19
2 February 2000

Conference Moderator:
LTC Dale G. Dunn
Department of Veterinary Pathology
Armed Forces Institute of Pathology
Washington, DC 20306-6000
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Case I - 99N185 (AFIP 2694824)
Signalment: 9-week-old, springer spaniel, female, canine
History: On presentation, the puppy had a large central corneal opacity associated with cords of tissue running from the iris collarette to the posterior cornea. The density of the opacity precluded visualization of the posterior aspect of the globe.
Gross Pathology: All non-ocular tissues were normal. The left eye had an asymmetric white opacity of the cornea and lens.
Case 19-1. Eye. Pigmented strands of uveal tissue extend from the iris and ciliary body to the cornea.
Laboratory Results: none.
Histopathology of brain, intestine, kidney, spleen, liver, heart, pancreas, lungs, ovary, uterus, and thyroid gland were within normal limits. Both eyes displayed indications of cataracts along the posterior aspect of the lens.
Histosections of the left eye revealed uveal cords of pigmented cells, spindle cells, and blood vessels stretching between the iris and the posterior cornea. Descemet's membrane and the corneal endothelium were disrupted at the site of adhesion. Radiating from these sites was often a multicellular, partially pigmented, variably vascularized membrane of spindle cells, pigmented cells, blood vessels, and glassy collagen similar to Descemet's membrane. At different sites along the cornea, there was localized disruption of Descemet's membrane with endothelial-like cells between it and the posterior stroma.
Most of the posterior stroma was within normal limits. The anterior stroma exhibited disorganized lamellae with apparent edema and some vascularization centrally. A zone of poor epithelial attachment was present centrally and was associated with membrane thickening, mineralization, and fragmentation. Some of the fragmented basement membrane was entrapped in the most superficial stroma, indicating band keratopathy.
Contributor's Diagnoses and Comments:
1) Anterior segment dysgenesis consistent with Peters' anomaly.
2) Superficial corneal edema and vascular invasion.
3) Posterior nuclear cataract.
Peters' anomaly is a developmental defect of the anterior segment, resulting in cords of uveal tissue adhering to the periphery of a central corneal leukoma. Histopathologically, Descemet's membrane and the corneal endothelium are reduced or absent in the area of the corneal opacification. The corneal stroma in this area is thin and hypercellular, and Bowman's layer may be absent. Approximately 80% of reported Peters' anomaly cases are bilateral. Glaucoma is frequently associated.
Normally corneal endothelium, corneal stroma, iris, and the iridocorneal angle arise from sequential waves of neural crest cells. Embryologically, a defect of neural crest migration is thought to result in Peters' anomaly. The abnormality is thought to be a heterogeneous defect but it may be associated with Pax6, which is a transcription factor in the "paired-box containing" gene family.
AFIP Diagnoses:
1. Eye: Incomplete Descemet's membrane and loss of corneal endothelium, central, focally extensive, with pigmented and vascularized iridokeratic cords, springer spaniel, canine.
2. Eye, cornea: Superficial edema and vascularization.
3. Eye, lens: Cataract.
4. Eye, retina: Dysplasia, focal (not present in all sections).
Conference Note: Arriving at a consensus on the morphologic diagnosis for this case became immediately problematic for conference participants because descriptions of both Peters' anomaly in the human medical literature and persistent pupillary membrane (PPM) in the veterinary medical literature are essentially identical. This conflict prompted a more descriptive approach to the morphologic diagnosis. However, conference participants did agree that using the current definitions either designation could apply in this case. We reviewed this case in consultation with the Department of Ophthalmic Pathology. They, understandably, favored a diagnosis of Peters' anomaly, stating that in humans PPM does not attach to the cornea. However, they conceded that this opinion does not fully account for the presence of the small blood vessels (that could be of pupillary membrane origin) evident within the iridokeratic cords. Reconciling these terminology differences is beyond the scope of this conference and awaits a review and revision of the subject in the veterinary medical literature.
Contributor: University of Wisconsin, School of Veterinary Medicine, Department of Pathobiological Sciences, 2015 Linden Drive West Madison, WI 53706.
1. Churchill AJ, Booth AP, Anwar R, Markham AF: Pax6 is normal in most cases of Peters' anomaly. Eye 12:299-303, 1998
2. Nakanishi J, Brown SJ: The histopathology and ultrastructure of congenital, central corneal opacity (Peters' anomaly). Am Journ of Ophthal 72(4):801-812, 1971
3. Spencer WH: Ophthalmologic Pathology, 4th ed., pp. 170-173. WB Saunders, Philadelphia, PA, 1996
4. Wilcock BP: The Eye and Ear. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 449-450. Academic Pres, San Diego, CA, 1993
5. Williams DL: A comparative approach to anterior segment dysgenesis. Eye 7:607-616, 1993
Case II -94N236 (AFIP 2500585)
Signalment: 8-year-old, Siberian husky, spayed-female canine.
History: An icteric dog with mucosal petechia was presented. Anemia and thrombocytopenia determined to be autoimmune were treated with steroids, but the dog developed respiratory distress and was euthanized. Hemorrhages were noted on the iris.
Gross Pathology: Icterus and pulmonary vascular thrombosis were found. The eyes were fixed in Bouin's solution.

Contributor's Diagnoses and Comments:
1. Iridal hemorrhage
2. Goniodysgenesis, Siberian husky, canine
Glaucoma is a common cause of ocular pain and vision loss in animals. Primary glaucoma in dogs usually occurs in globes with an abnormal morphological development of the iridocorneal angle structures known as goniodysgenesis or mesodermal dysgenesis. Affected dogs have abnormal angle morphology prior to the development of glaucoma. Furthermore, only a small percentage of dogs with goniodysgenesis will develop glaucoma. If one eye becomes affected, the risk of disease in the other eye is high, but the time of onset is unpredictable. The eye submitted has the typical features of goniodysgenesis in a normotensive eye from a commonly affected breed. Rather than a series of primary pectinate ligaments, this eye has a solid sheet of uveal tissue connecting the iris base and the end of Descemet's membrane. The terminus of Descemet's membrane also shows abnormal thickening and branching, and extends deep into the ciliary body. The iris hemorrhage was likely due to thrombocytopenia.
AFIP Diagnoses:
1. Eye: Peripheral anterior segment dysgenesis (goniodysgenesis), Siberian husky, canine.
2. Eye, iris: Hemorrhage, focally extensive.
3. Eye: Anterior uveitis, plasmacytic and lymphocytic, diffuse, mild.
4. Eye, ora ciliaris retinae: Cystic degeneration.

Conference Note: This case was also reviewed in consultation with the Department of Ophthalmic Pathology. As discussed in the conference note for case I of this conference, improper formation of the anterior segment results in a variety of histologic lesions. In humans, peripheral anterior segment dysgenesis consists of a spectrum of defects known by their eponyms. Axenfeld's anomaly consists of peripheral defects including a prominent Schwalbe's line, with or without strands, between the iris and cornea. Rieger's anomaly consists of these peripheral changes complicated by iridal defects. Rieger's syndrome encompasses these changes and non-ocular defects.
The Department of Ophthalmic Pathology preferred a diagnosis of Axenfeld's anomaly for this case. However, strict adherence to medical pathology terminology is difficult, given the lack of a Schwalbe's line in nonprimate species. Conference participants favored the less specific diagnosis of peripheral anterior segment dysgenesis and considered goniodysgenesis an accurate and reasonable descriptive synonym for this histologic change. For an excellent review of anterior segment dysgenesis see reference 5 below.
Contributor: University of Wisconsin-Madison, Department of Pathological Sciences, School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706-1102.
1. Gwin RM: Current Concepts in Small Animal Glaucoma: Recognition and Treatment. Vet Clin of N Am/ Sm An Pract 10:357-376, 1980
2. Martin CL: Scanning Electron Microscope Examination of Selected Canine Iridocorneal Angle Abnormalities. Scan Elect Mic Ex 11:300-306, 1975
3. Spencer WH: Ophthalmologic Pathology, 4th ed., pp. 170-173. WB Saunders, Philadelphia, PA, 1996
4. Wilcock BP: The Eye and Ear. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, vol. 1, 4th ed., pp. 449-450. Academic Pres, San Diego, CA, 1993
5. Williams DL: A comparative approach to anterior segment dysgenesis. Eye; 7:607-616, 1993
Case III - Eli Lilly and Co, Rat A, X6,100 and Eli Lilly and Co, Rat A, X10,200Rat A (AFIP2686560 )
Signalment: F344 rat, female, approximately 2 years of age
History: The nodule was an incidental finding at necropsy. After histologic examination, remaining formalin-fixed tissue was processed for transmission electron microscopy (TEM).
Gross Pathology: At necropsy, the rat had a partially ulcerated skin nodule, approximately 1.3 x 0.5 cm, on the pinna. The nodule was firm and white on section.

Contributor's Diagnosis and Comments: Amelanotic melanoma
Histologic sections (limited slides submitted).

Skin (pinna). The dermis is expanded by a densely cellular neoplasm composed of closely-packed spindle cells with indistinct cell borders, scant amounts of cytoplasm and oval nuclei, generally with 1-3 inconspicuous nucleoli. Zero-1 mitotic figures are noted per 40x field. The neoplastic cells are arranged in interwoven bundles, and whorls, sometimes forming a "storiform" pattern. The neoplastic cells abut the epidermis and surround the auricular cartilage. The overlying epidermis is covered by serocellular crusts, is attenuated or partially ulcerated in some areas, and has multifocal areas of hyperplasia with formation of small rete pegs that extend into the neoplasm.
Immunohistochemical staining (no slides submitted).

Neoplastic cells had minimal to slight diffuse cytoplasmic staining and more densely-granular, nuclear staining for S-100 protein. The location, histologic appearance and positive S-100 reactivity of the neoplasm were suggestive of amelanotic melanoma. This diagnosis was confirmed by identification of premelanosomes in the neoplastic cells via TEM. Findings in this case are similar to those described by Nakashima, et al.
Four stages of melanosomes have been identified by Fitzpatrick et al (Yoshimoti, 1991). However, in albino rats, normal melanocytes usually contain only premelanosomes (stage II melanosomes) which cannot produce melanin. Spontaneous melanomas reported previously in F344 rats had characteristic premelanosomes, in contrast to chemically-induced uveal melanomas which also contained stage III and IV (pigmented) melanosomes (Yoshitomi, 1993). The morphologic features of the neoplasm presented here are consistent with a spontaneous, aural, amelanotic melanoma.
Case 19-3. Rat A, 6100x
This electron micrograph contains portions of multiple, interdigitating spindle cells, 2 with visible nuclei. The cells have ovoid nuclei with dispersed chromatin and abundant cytoplasmic organelles, and are joined by desmosomes. The most prominent organelles are multiple, ovoid, approximately 0.3 x1.0 (single membrane-bound structures, with multiple internal membranous filaments arranged in parallel to the long axis [premelanosomes (stage II melanosomes)]. Other cytoplasmic organelles are present in low number and include rough endoplasmic reticulum (RER), mitochondria and multiple membrane-bound vacuoles with scant contents (most likely dilated RER).
Case 19-3. Rat A, 10200x
This electron micrograph illustrates the finer detail of the premelanosomes as well as the multiple desmosomes between the neoplastic cells, and absence of a basal lamina.
AFIP Diagnosis: Pinna (per contributor): Spindle cells with numerous intracytoplasmic premelanosomes and absence of melanin, Fisher 344 rat, rodent.
Conference Note: This case was chosen for the Wednesday Slide Conference because it provided the opportunity to review, describe and interpret an electron micrograph. Conference participants readily identified the spindle cells as having features suggesting melanocytic origin. Given signalment alone, they suspected amelanotic melanoma but few were willing to make a definitive diagnosis based on the few cells evident in the micrograph. With the additional history of a mass on the pinna, most were confident of the diagnosis.
The differential diagnosis of this case includes schwannoma. Schwann cells in both rats and humans can contain melanosomes, but they are usually surrounded by a basal lamina. Although only two cells are present in the electron micrographs, a basal lamina is not observed in these photos.
Although amelanotic melanomas are considered rare in albino rats (such as the Fisher 344), they are relatively common in pigmented varieties. Along with the pinna, other common sites for melanomas in rats include the uvea, tail, and genitalia.
Contributor: Lilly Research Laboratories, PO Box 708, Greenfield, IN 46140.
1. Nakashima N, Mitsumori K, Maita K, Shirasu Y: Amelanotic melanocytic tumors of the pinna in six F344 rats. J Vet Med Sci 53(2):291-296, 1991
2. Yoshitomi K, Boorman G: Palpebral amelanotic melanomas in F344 rats. Vet Pathol 30(3):280-286, 1993
3. Yoshitomi K, Boorman G: Spontaneous amelanotic melanomas of the uveal tract in F344 rats. Vet Pathol 28(5):403-409, 1991
Case IV - 19361 (H&E); 19361 BH (Brown-Hopps Gram)(AFIP 2681374)
Signalment: 2-month-old male New Zealand white rabbit (Oryctolagus cuniculus).
History: This rabbit was purchased from a supplier of laboratory rabbits and acclimated in the recipient animal facility for a few days. It was in excellent condition and had no signs of disease. The investigator withheld food for 24 hours in preparation for surgery. The rabbit was found dead in its cage the morning of the scheduled surgery.
Gross Pathology: At necropsy, the perineum, thighs and hocks had tan, watery fecal staining. The serosa of the cecum had a few scattered petechiae and the wall appeared thickened. The cecal lumen was » 1.0 to 1.5 cm in diameter and contained pink, gelatinous mucoid material; the mucosa was
roughened and dark pink. The colon and the rectum contained minimal fluid feces and had normal appearing mucosae. Touch preparations were made for Gram stain of contents at three levels: cecum, mid colon and rectum. The liver, thymus and tracheal mucosa were reddened.
Laboratory Results:
  Aerobic Bacterial Cultures:
Conjunctiva- negative culture
Nasal swab: gamma Streptococcus, Staphylococcus epidermidis, Corynebacterium sp. (non-pathogenic)
Small intestine: negative culture
Cecum: Enterococcus, Streptococcus viridans, Bacillus sp.
Colon- Enterococcus, Streptococcus viridans, Corynebacterium sp. (non-pathogenic)
Anaerobic Bacterial Cultures: None
 Gram stains of contents of large intestine:
Cecum: Mucoid contents contained massive numbers of large C-shaped and coiled Gram positive rods compatible with Clostridium spiroforme (see 2 X 2 slides from this case and those of Gram stained cecal contents from a normal control rabbit from the same supplier).
Colon: Few large Gram positive rods compatible with Clostridium spiroforme
Rectum: Few large Gram positive rods compatible with Clostridium spiroforme

Contributor's Diagnosis and Comments: Necrotizing typhlitis, peracute, severe and diffuse, compatible with Clostridium spiroforme mediated enterotoxemia, probably provoked by extended withholding of food.

Additional findings in this case included: (i) mild necrotizing enteritis in the sacculus rotunda and appendix, (ii) mild acute mesenteric lymphadenitis, (iii) severe myeloid depletion of bone marrow and spleen, (iv) severe congestion of viscera, and (v) moderate neuronal shrinkage and basophilia in Ammon's horn of the dentate gyrus consistent with anoxia. All of these changes were thought to be directly or indirectly associated with the C. spiroforme mediated enterotoxemia.
Specimens of intestine were fixed intact in alcoholic formalin (10% formalin in 70% ethanol) to limit autolysis of the mucosa and allow visualization in sections of the undisturbed intestinal contents-mucosal relationships. The severe necrotizing lesions in the cecum and similar lesions in the sacculus and appendix, coupled with the presence of massive numbers of large curved or coiled Gram positive bacteria typical of C. spiroforme in the contents of the affected bowel, provided very strong presumptive evidence of C. spiroforme mediated enterotoxemia as the central disease process in this case. Definitive proof would have required culture of C. spiroforme and demonstration of C. spiroforme enterotoxin in the intestinal contents by biochemical, immunological, cytotoxicity assay or other methods, not routinely available in most labs. Enteritides due to aerobic bacteria were ruled out by negative cultures. There was no morphologic evidence of Clostridium pilifome infection in the intestine or liver. Additionally, current health surveillance testing had shown the stock to be serologically negative for C. piliforme and rotavirus, negative for Lawsonia intracellularis by PCR testing and negative for other common nonintestinal pathogens of rabbits.
C. spiroforme is considered one of the leading causes of enteropathy in weanling rabbits. Overgrowth of C. spiroforme and other toxin producing clostridia in the cecum (mainly) of rabbits has been associated with numerous factors, including weaning, dietary change and antibiotic administration. The extended withholding of food and the young age (perhaps limited GI flora?) of this rabbit probably were major contributing factors to the massive overgrowth of C. spiroforme in the cecum, resulting in a fatal case with all of the characteristic features of peracute C. spiroforme mediated enterotoxemia.
Much is known about the molecular pathogenesis of enterotoxemia due to C. spiroforme. The toxin is a protein that consists of two functional domains, A and B. The B domain binds to receptors on enterocytes and delivers the toxic A moiety into the cytosol. Toxin A is an ADP (adenosine 5'-diphosphate)-ribosyltransferase that removes nicotinamide from a ribose of nicotine adenine dinucleotide and attaches the ribose to Rho, small GTP (guanosine 5'-triphosphate)-binding proteins known as Rho GTPases involved in the regulation of actin filament assembly. Ribosylation functionally inactivates Rho and results in depolymerization of filamentous (F) actin which, in turn, results in loss of cell polarity and adhesion, leading to rounding and necrosis of enterocytes. Function of the Rho GTPases is of critical importance as they regulate a plethora of other cell functions, including cytokinesis, phagocytic NADPH, serum- and growth factor-mediated signaling, nuclear signaling and induction of apoptosis. Based on studies of similar toxins A and B of Clostridium difficile which have been studied far more extensively, toxins A and B of C. spiroforme may inactivate Rho through other chemical processes, and have numerous other deleterious effects such as inducing secretion and fluid accumulation, chemotaxis, cytokine and chemokine secretion, and many others (see Fasano, 1999; Guerrant et al., 1999).
AFIP Diagnosis: Cecum: Typhlitis, erosive, acute, diffuse, severe, with loss of glands and numerous luminal coiled Gram positive bacilli, New Zealand white rabbit (Oryctolagus cuniculus), lagomorph.
Conference Note: Conference participants agreed with the contributor that this case probably represents an example of enterotoxemia resulting from Clostridium spiroforme infection. While the morphology of this organism is fairly distinctive, participants reaffirmed the need for bacterial culture and toxin detection for definitive diagnosis. The contributor has provided an excellent review of the clinical and pathological findings as well as the pathogenesis and differential diagnosis for enterotoxemia caused by C. spiroforme.
Contributor: Department of Comparative Medicine, University of Alabama at Birmingham, Birmingham, AL 35243-0019.
1. Aktories K: Identification of the catalytic site of clostridial ADP-riboslytransferases. Adv Exper Med Biol 419:53-60, 1997
2. Bouquet P, Gill DM: Modulation of cell functions by ADP-ribosylating bacterial toxins., In: Sourcebook of Bacterial Protein Toxins. eds. Alouf JE, Freer JH, pp. 23-44. Academic Press: San Diego, CA, 1991
3. Butt MT, Papendick RE, Carbone LG, Quimby FW: A cytotoxicity assay for Clostridium spiroforme enterotoxin in cecal fluid of rabbits. Lab Anim Sci 44:52-54, 1994
4. Carman RJ, Evans RH: Experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs. Lab Anim Sci 34:443-452, 1984
5. DeLong D, Manning PJ: Bacterial diseases, III. Enterotoxemia. In: The Biology of the Laboratory Rabbit, eds. Manning PJ, Ringler DH, Newcomer CE, 2nd ed., pp. 140-143. Academic Press, San Diego, CA, 1994
6. Fasano A: Cellular microbiology: can we learn cell physiology from microorganisms?: Am J Physiol 276:C765-C776, 1999
7. Guerrant RL, Steiner TS, Lima AAM, Bobak DA: How intestinal bacteria cause disease. J Infect Dis 179(Suppl 2):S331-337, 1999
8. Holmes HT, Sonn RJ, Patton NM: Isolation of Clostridium spiroforme from rabbits. Lab Anim Sci 38:167-168, 1988
9. Popoff MR, Milward FW, Bancillon B, Boquet P: Purification of the Clostridium spiroforme binary toxin and activity of the toxin on HEp-2 cells. Infect Immun 57:2462-2469, 1989
10. Rappuoli R, Pizza M: Structure and evolutionary aspects of ADP-ribosylating toxins., In: Sourcebook of Bacterial Protein Toxins. eds. Alouf JE, Freer JH, pp.1-21. Academic Press, San Diego, CA, 1991
11. Sears CL, Kaper JB: Enteric bacterial toxins: Mechanisms of action and linkage to intestinal secretion. Microbiol Rev. 60:167-215, 1996
12. Simpson LL, Stiles BG, Zepeda H, Wilkins TD: Production by Clostridium spiroforme of an iota like toxin that possesses mono(ADP-ribosyl)transferase activity: Identification of a novel class of ADP-ribosyltransferases. Infect Immun 57:256-261, 1989
13. Yonushonis WP, Roy MJ, Carman RJ, Sims RE: Diagnosis of spontaneous Clostridium spiroforme iota enterotoxemia in a barrier rabbit breeding colony. Lab Anim Sci 37:69-71, 1987
J Scot Estep, DVM
Captain, United States Army
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202) 782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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