AFIP Wednesday Slide Conference - No. 30

27 May 1998

Conference Moderator:
LTC Thomas P. Lipscomb, Diplomate, ACVP
Division of Veterinary Pathology
Armed Forces Institute of Pathology
Washington, D.C. 20306

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Case I - S-51-96 (AFIP 2593311)

Signalment: Free-ranging, male, pink river dolphin (Inia geoffrensis).

History: This animal was captured in the course of a radio-tagging field study conducted in Lago Mamiraua, Brazil.

Gross Pathology: A heavily vascularized, multinodular, pedunculated mass was attached to the right eye. It was firmly adhered to, and appeared to arise from, the conjunctiva of the dorsal palpebrum as well as the medial and lateral canthus.

Contributor's Diagnosis and Comments: Conjunctiva: Conjunctivitis, proliferative, chronic-active to granulomatous, diffuse, marked, with mucosal hyperplasia and intralesional fungal elements compatible with Rhinosporidium seeberi.
The polyploid conjunctival lesions seen in this dolphin are "classic" examples of an infection with Rhinosporidium seeberi. Although a number of different blocks were used for glass slide recuts, all slides demonstrate the developmental stages of R. seeberi. These are readily identified by specific histologic features. These are:

1. Trophocyte (juvenile sporangia)
- averages 10-100 microns
- has a 2-3 micron unicellular wall
- each has a central nucleus with prominent nucleolus
- granular to flocculent cytoplasm
- no mature endospores
2. Intermediate sporangia
- are larger than trophocytes
- these lack a nucleus
- have thicker, bilamellar walls
3. Mature sporangia
- hallmark of R. seeberi infection
- average 100-300 microns diameter
- "zonation" of mature and immature endospores
- mature endospores have "eosinophilic globular bodies"

Little is known about the epidemiology of this organism. Although generally considered to be a fungus, its taxonomy is still uncertain and it has yet to be cultivated on synthetic media. R. seeberi typically induces the formation of chronic inflammatory nasal polyps. Conjunctival lesions have been reported, but these are far less common than respiratory lesions.

This organism has been studied most intensively in man, although animal cases have been documented and described in horses, mules, cattle, dogs, a goose, 2 ducks and, most recently, in 41 swans from a lake in central Florida. This outbreak lends support to the hypothesis that R. seeberi may be an aquatic organism.

Case 30-1. Skin. A mature sporangium of Rhinospordium seeberi is discharging some of its endospores to surface of the skin. A small portion of an immature sporangium is at the edge. It contains only floccular eosinophilic material. 20X

AFIP Diagnosis: Stratified squamous epithelium and loose connective tissue: Inflammatory polyp, with moderate chronic-active inflammation, multifocal hemorrhage, epithelial necrosis, and multiple fungal sporangia, pink river dolphin (Inia geoffrensis), cetacean, etiology consistent with Rhinosporidium seeberi.

Conference Note: Based only on the morphology of the infecting organism, the differential diagnosis might include coccidioidomycosis and adiasporomycosis. However, the spherules of Coccidioides immitis are generally smaller than rhinosporidial sporangia, and they contain endospores that are generally the same size and shape throughout the spherule. Chrysosporium parvum, the etiologic agent of adiasporomycosis, is larger than Rhinosporodium, has a thicker wall, and does not reproduce by endosporulation.

Rhinosporidiosis occurs sporadically worldwide, but is hyperendemic in India, Sri Lanka, and Southeast Asia.1,2 Infection is associated epidemiologically with rural and aquatic environments. A recent study from India suggests that Rhinosporidium seeberi is a form of the cyanobacterium Microcystis aeruginosa, which was isolated from water samples in which human patients with rhinosporidiosis were bathing.8

Contributor: Wildlife Conservation Society, Department of Pathology, 185th St. and Southern Blvd., Bronx, NY 10460

1. Chandler FW, Kaplan W, Ajello L: Color Atlas and Text of the Histopathology of Mycotic Diseases. Year Book Medical Publishers, Inc. Chicago. pp. 109-111, 1980.
2. Chandler FW, Watts JC: Pathologic Diagnosis of Fungal Infections. ASCP Press, Chicago. pp. 27-33, 1987.
3. Cheville NF: Ultrastructural Pathology. An Introduction to Interpretation. Iowa State University Press. Ames, IA. pp. 775-776, 1994.
4. Gaines JJ, Clay JR, Chandler FW, Powell ME, Sheffield PA, Keller III AP: Rhinosporidiosis: three domestic cases. Southern Medical Journal 89(1):65-67, 1996.
5. Kennedy FA, Buggage RR, Ajello L: Rhinosporidiosis: A description of an unprecedented outbreak in captive swans (Cygnus spp.) and a proposal for revision of the ontogenic nomenclature of Rhinosporidium seeberi. Journal of Medical and Veterinary Mycology 33:157-165, 1995.
6. Kwon-Chung KJ: Phylogenetic spectrum of fungi that are pathogenic to humans. Clinical Infectious Diseases 19 (Suppl 1): S1-7, 1994.
7. Levy MG, Meuten DJ, Breitschwerdt EB: Cultivation of Rhinosporidium seeberi in vitro: interaction with epithelial cells. Science 234:474-476, 1986.
8. Ahluwalia KB, Maheshwari N, Deka RC: Rhinosporidiosis: a study that resolves etiologic controversies. Am J Rhino 11(6):479-483, 1997.

International Veterinary Pathology Slide Bank:
Laser disc frame #6637, 6638, 6639, 8246, 4471, 14472, 14473


Case II - 94-114 (AFIP 2453724)

Signalment: 8-week-old, female, New Zealand White, rabbit.

History: Incidental finding in a rabbit infected with Campylobacter jejuni.

Gross Pathology: None.

Contributor's Diagnosis and Comments: Kidney: Nephritis, interstitial, chronic, moderate, with gram positive intraepithelial protozoal organisms - Etiology consistent with Encephalitozoon cuniculi.

Encephalitozoon cuniculi is an occasional, usually asymptomatic, parasite of rabbits. The primary importance of the organism in rabbits is interference with the interpretation of experimental data. In other species (dogs, cats, and wild carnivores) the organism causes clinical, often fatal encephalitis and nephritis.

The organisms are most commonly found in the renal tubular epithelial cells and capillary endothelial cells within the central nervous system.

Differentiating Encephalitozoon cuniculi from toxoplasmosis can be accomplished a number of ways. Encephalitozoon stains poorly on H&E, is gram positive, and its spores are birefringent; Toxoplasma stains well on H&E, stains poorly with Gram stains, and is not birefringent.
Case 30-2a. Kidney. Tubule whose lumen is filled with necrotic epithelial cells that contain numerous but vague eosinophilic organisms in the cytoplasm. 40X
Case 30-2b. Kidney. Numerous microsporidian organisms (Encephalitozoon cuniculi) in a degenerating tubule. Gram. 40X

AFIP Diagnosis: Kidney: Nephritis, tubulointerstitial, acute to chronic, multifocal, moderate, with tubular dilatation, tubular epithelial necrosis, and intracellular and extracellular protozoa, New Zealand white rabbit, lagomorph.

Conference Note: Transmission of Encephalitozoon cuniculi, a microsporidian, is primarily via ingestion of urine containing the infective spores. Transplacental infection has also been reported.3 In addition to the susceptible species listed above, infection occurs in rats, mice, guinea pigs, hamsters, and humans.3 At least three strains of E. cuniculi have been identified based on host specificity and other criteria.4

Encephalitozoon cuniculi infection of mice is used as a model of human microsporidiosis. Mouse strains differ greatly in their susceptibility to infection, with C57BL/6, DBA/1, and 129J being highly susceptible, and BABL/c, A/J, and SJL strains being relatively resistant.4 Athymic (nu/nu) mice experience high mortality with infection.

Contributor: Naval Medical Research Institute, Pathobiology Division, 8901 Wisconsin Avenue, Bethesda, MD 20889-5607

1. Szabo JR, Shadduck JA: Experimental encephalitozoonosis in neonatal dogs. Vet Pathol 24:99-108, 1987.
2. Cutlip RC, Beall CW: Encephalitozoonosis in arctic lemmings. Lab Anim Sci 39(4):331-333, 1989.
3. Soulsby EJL: Helminths, Arthropods and Protozoa of Domesticated Animals, 7th edition, Lea & Febiger, Philadelphia, pp. 742-743, 1982.
4. Baker DG: Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clinical Microbiology Reviews 11(2):231-266, 1998.

International Veterinary Pathology Slide Bank:
Laser disc frame #5278-5281, 19463.


Case III - Mississippi State University (AFIP 2376327)

Signalment: Adult, female, cottontail rabbit (Sylvilagus floridanus)

History: This animal was found alive but weak, depressed, thin, and easily captured.

Gross Pathology: The animal was emaciated, flea infested, and had several ticks. The peritoneal cavity contained approximately 10 ml of serous fluid. The spleen was 2x2x6 cm, and multiple disseminated pinpoint to 1 mm white foci were visible on the capsular and cut surfaces. The liver had similar foci. Mesenteric, hilar and mediastinal lymph nodes were enlarged, gray, and friable. Several tapeworm cysts were noted in the peritoneal cavity and attached to the liver and pleura.

Laboratory Results: Francisella tularensis was isolated.

Contributor's Diagnosis and Comments: Disseminated multifocal and coalescing necrotizing splenitis.

This case represents a classic case of tularemia. Care should be taken by prosectors as this condition is zoonotic.
Case 30-3. Spleen. There is extensive liquefactive necrosis of paranchyma with vague, basophilic, intralesional bacterial colonies (center). 20X
AFIP Diagnosis: Spleen: Splenitis, necrotizing, acute to subacute, multifocal to coalescing, severe, with necrotizing vasculitis, fibrin thrombi, and numerous colonies of coccobacilli, cottontail rabbit (Sylvilagus floridanus), lagomorph.

Conference Note: Gram stains demonstrated that the coccobacilli are gram-negative.

Tularemia (deer fly fever, rabbit fever) is a zoonotic disease with worldwide distribution, affecting more than 100 species of wild and domestic mammals, birds, fish, and reptiles. It is primarily a disease of wild rabbits and rodents and -shares many key features with endemic plague, with which it was originally confused in a 1911 outbreak in ground squirrels in Tulare County, California.

There are two antigenically similar strains of F. tularensis:

1. F. tularensis subsp. tularensis (Type A) occurs only in North America, where it is the most frequently isolated strain (70% of human cases). It is associated with tick borne tularemia in rabbits, and produces the classic disease in humans.
2. F. tularensis subsp. palaearctica (Type B) occurs throughout the world (except Australia and Antarctica), and is less virulent than Type A. It is associated with mosquitoes and rodents, and is frequently linked to waterborne disease of rodents, particularly beavers and muskrats.

Transmission of the disease may occur by a variety of routes including direct contact of the organism with intact or abraded skin or mucous membranes, ingestion or inhalation of the organism, or by percutaneous inoculation via arthropod vectors. As few as 10 bacilli may induce disease when inhaled or injected, whereas a much larger dose is required for oral infection.

Ticks are reported most frequently as the source of human infection in the United States, followed by rabbits. Two seasonal peaks are associated with tularemia: one during tick season, the other during the hunting season, associated with contact with infected rabbits. Dermacentor variabilis (dog tick), D. andersoni (wood tick), and Amblyomma americanum (lone star tick) are considered the most important tick vectors in North America. Both transstadial and transovarian passage of F. tularensis have been documented, making the tick both a vector and a reservoir of infection. The deerfly, Chrysops discalis, is also an important vector in North America.

Clinical signs are those of an acute septicemia and vary with the route of infection, the strain of the organism, and the species involved. Rabbits and rodents are often found dead without premonitory signs. In cats, tularemia is associated with nonspecific clinical signs, including pyrexia, anorexia, lethargy, lymphadenopathy, oral ulcers, hepatomegaly, and icterus.2

Contributor: Mississippi State University, College of Veterinary Medicine, Mississippi State, MS 39762

1. Davidson WR, Nettles VF: Field Manual of Wildlife Diseases in the Southeastern United States. Southeastern Cooperative Wildlife Disease Study, pp 212-215, 1988.
2. Woods JP, Crystal MA, Morton RJ, Panciera RJ: Tularemia in two cats. JAVMA 212(1):81-83, 1998.
3. Valli VEO: The hematopoietic system. In: Pathology of Domestic Animals, 4th edition, Jubb KVF, Kennedy PC, Palmer N (eds.), Academic Press, Inc, vol. 3, pp. 244-245, 1993.

International Veterinary Pathology Slide Bank:
Laser disc frame #3476, 3477, 5307, 5308, 11121-11124, 22253.


Case IV - 90-4468 (AFIP 2327342)

Signalment: 2-year-old, male, Doberman Pinscher, canine, named "Mr. Blue".

History: This dog had a history of chronic keratoconjunctivitis and generalized bilaterally symmetrical hair loss.

Gross Pathology: Bilaterally symmetrical hair loss.

Laboratory Results: Antinuclear antibody: 1:20
Baseline T4: 0.2 (normal 1.0-4.0)

Contributor's Diagnoses and Comments:

Haired skin: Diffuse superficial and follicular orthokeratotic hyperkeratosis
Multifocal follicular atrophy with melanin clumping
Multifocal, perifollicular melanophage accumulation
Mild superficial perivascular hyperplastic dermatitis

Condition: Color mutant alopecia.

These sections demonstrate the classic histologic features of color mutant alopecia in the dog: diffuse superficial and follicular orthokeratotic hyperkeratosis; dilated keratin-filled follicles; multiple melanin aggregates within hair shafts and bulbs; occasional fractured hairs; diffuse, moderate adnexal atrophy; and scattered, generally perifollicular, deep dermal and pannicular aggregates of melanophages. In this case, there is a minimal superficial perivascular dermatitis with mild epidermal hyperplasia. Most sections have one or more anagen hair follicles, though many follicles are atrophied. Some sections contain intrafollicular accumulations of inflammatory cells. Arrector pili muscles are present, but not enlarged or vacuolated. The low baseline T4 level might indicate, but is not diagnostic for, hypothyroidism. There are no histologic changes to suggest hypothyroidism and it is unknown if the dog was on thyroid medication at the time of biopsy. Besides the blue Doberman, this condition can be seen in fawn Irish Setters, red and fawn Dobermans, and other "blue" varieties of various breeds.
Case 30-4. Skin. There is hair follicle atrophy with clumping of melanin in one follicle accompanied by mild intrafollicular hyperkeratosis. 4X
AFIP Diagnosis: Haired skin: Follicular atrophy, ectasia, and hyperkeratosis, diffuse, moderate, with intrafollicular melanin clumping, peribulbar melanophages, and mild multifocal superficial lymphoplasmacytic and eosinophilic dermatitis, Doberman Pinscher, canine.

Conference Note: This hereditary syndrome is associated with a color-dilution gene, but it is not known if the gene is directly responsible for initiating the skin disease or if a linked gene codes for the associated follicular changes.2

Clinically, this disease is characterized by a gradual onset of a dry, dull, brittle, poor-quality hair coat. Hair shafts break, and regrowth is often poor. Follicular papules and comedones may develop, and chronic cases may exhibit hyperpigmentation.

The clinical differential diagnosis of color mutant alopecia should include other generalized atrophic or dysplastic diseases affecting the hair follicle including hypothyroidism, hyperadrenocorticism, canine follicular dysplasia, and acquired pattern alopecia.2

Contributor: Department of Pathology, Cornell University, Ithaca, NY 14853

1. Brignac MM, Foil CS, Al-Bagdadi FAK, Kreeger J: Microscopy of color mutant alopecia. Ann Meet Am Acad Vet Dermatol and Am Coll Vet Dermatol., pp. 14-15, 1988.
2. Gross TL, Ihrke PJ, Walder EJ: Veterinary Dermatopathology. A macroscopic and microscopic evaluation of canine and feline skin disease. Mosby Year Book, St. Louis, MO, pp. 298-301, 1992.
3. Scott DW, Miller Jr WH, Griffin CE: Muller & Kirk's Small Animal Dermatology, 5th edition, W.B. Saunders Company, Philadelphia, pp. 777-779, 1995.

International Veterinary Pathology Slide Bank:
Laser disc frame #11787, 11856, 11857, 13848-13851.

Terrell W. Blanchard
Major, VC, USA
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615

* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.

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