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CASE I – N96-4286 (AFIP 2597323)
Signalment: 2-month-old female quarterhorse
History: The filly was purchased at a horse sale and developed watery diarrhea. Therapy with kaolin pectin, intravenous fluids, and Excenel was performed with no clinical improvement. The diarrhea was green and watery. The filly was euthanized 10 days after initial purchase. She had lost about 60 pounds of body weight.
Gross Pathology: The submitting veterinarian described the small intestine as being diffusely thickened with a velvety appearance of the mucosa. The colon and rectum contained only green watery material. A three-inch segment of thick-walled intestine was tied at either end and submitted in formalin. The serosal surface exhibited a cerebriform appearance due to thick corrugations of the mucosa. Small mucosal hemorrhages were present.
Laboratory Results: Fecal flotation and blood work were within normal limits.
Contributor’s Diagnosis and Comment: Small intestine: Enteritis, diffuse, severe, proliferative, with intracellular bacteria in apical cytoplasm of mucosal epithelial cells.
Etiology: Lawsonia intracellularis-like bacterium
Intestinal adenomatosis is common in the pig, and has been reported in horses, rabbits, sheep, foxes, ferrets, guinea pigs, and hamsters. In this case, silver stains and electron microscopy helped to confirm the diagnosis. No bacterial cultures, in situ hybridization, or polymerase chain reaction assays were performed. A few coccidial oocysts are present in the histologic sections submitted. The normal villus architecture of the intestine is obliterated by severely hyperplastic crypt-like epithelial cells. The accompanying Warthin-Faulker silver stain shows myriad curved bacterial rods in the apical cytoplasm of mucosal epithelial cells.
AFIP Diagnoses: 1. Small intestine: Enteritis, proliferative, histiocytic and neutrophilic, diffuse, moderate, with villar blunting and fusion, crypt abscesses, and numerous intraepithelial argyrophilic curved bacilli, etiology consistent with Lawsonia intracellularis, quarterhorse, equine.
2. Small intestine: Abscess, submucosal, with plant material.
3. Small intestine, lamina propria: Coccidial organisms, few, etiology consistent with Eimeria spp.
Conference Comment: Recent reports indicate that Lawsonia intracellularis is the cause of the proliferative enteropathies observed in a variety of species.
The means by which Lawsonia intracellularis induces intestinal crypt cell hyperplasia in animals is unknown. Suggested mechanisms include mimicry of an intracellular or extracellular signal for cell division, and down-regulation of apoptosis. An increase in the number of bacteria precedes the onset of hyperplasia, indicating a close association between the two events.
Lesion development, however, appears to be dependent on the presence of a complex mixture of intestinal microbial flora. Germ-free or gnotobiotic piglets, with a simple intestinal flora, are resistant to infection by L. intracellularis; exposure fails to result in lesions and enterocyte colonization cannot be detected. However, addition of a more complex, but incomplete, intestinal flora results in infection and disease. The complex flora may modify pathogenicity by altering the intestinal reduction-oxidation potential or rate of enterocyte division.
Entry of bacteria into the cells appears to be an active process of uptake through the cell membrane, producing membrane-bound endocytic vacuoles. Soon after release from the vacuoles, bacteria begin to replicate free in the cytoplasm. Infected epithelial cells may then populate the neighboring area in a manner similar to that of normal epithelial replacement. The shedding of infected cells from the villus provides a mechanism by which the bacteria may colonize distal intestinal tissue.
Modification of the immune system by coinfection with other known pathogens, and mucous cell depletion causing a loss of the physical mucous barrier from the intestinal surface have been proposed as possible cofactors in the pathogenesis of proliferative enteropathies induced by L. intracellularis. Diet, microenvironmental factors, and the presence of essential cellular receptors are also thought to be important.
Contributor: University of Saskatchewan, Western College of Veterinary Medicine, 52 Campus Drive, Saskatoon, SK, S7N 5B4, Canada
References: 1. Cooper DM, Gebhart CJ: Comparative aspects of proliferative enteritis. J Am Vet Med Assoc 212(9):1446-1451, 1998
2. Duhamel GE, Wheeldon EB: Intestinal adenomatosis in a foal. Vet Pathol 19:447-450, 1982
3. Lavoie JP, Drolet R, Parsons D, Leguillette R, Sauvageau R, Shapiro J, Houle L, Hallé G, Gebhart CJ: Equine proliferative enteropathy: a cause of weight loss, colic, diarrhoea and hypoproteinaemia in foals on three breeding farms in Canada. Eq Vet J 32(5):418-425, 2000
4. Lawson GHK, Gebhart CJ: Proliferative enteropathy. J Comp Path 122:77-100, 2000
5. Williams NM, Harrison LR, Gebhart CJ: Proliferative enteropathy in a foal caused by Lawsonia intracellularis-like bacterium. J Vet Diagn Invest 8:254-256, 1996
CASE II – E3202/99 (AFIP 2738552)
Signalment: 10-year-old female gazelle (Gazella dorcas)
History: Over several weeks, the gazelle developed a cutaneous lump near the left carpus. The fibrous mass was surgically removed and submitted for histological examination. The animal is doing well.
Gross Pathology: The specimen consisted of a 3.5 cm, bright green, ovoid mass with a lobular pattern surrounded by a fibrous capsule. The overlying haired skin was ulcerated.
Laboratory Results: The existence of starch granules and chloroplasts within developmental stages of the organism was proven by electron microscopy.
Contributor’s Diagnosis and Comment: Haired skin: Dermatitis, proliferative, granulomatous, chronic, severe with myriad intra- and extracellular spherical organisms, etiology consistent with Chlorella.
Since the overlying epithelium is not present in all sections, and the lesions here are of minor importance, they are not described. The mass consists of granulomatous inflammatory tissue. Between broad strands of fibrous tissue, there are large accumulations of macrophages, many of which appear as ghost cells within areas of necrosis. There are rare lymphocytes, plasma cells, and multinucleated giant cells. Variable degrees of fibroplasia are present within the inflammatory tissue. Countless spherical to slightly ovoid organisms are surrounded by granulomatous inflammation. The organisms measure 8–43 m m and represent various developmental stages. Among transitional stages, two main types can be distinguished: small bodies with thin membranes and basophilic granules that stain strongly by periodic acid-Schiff reaction, and larger bodies with thick membranes and a lucent, slightly eosinophilic, cloudy center. A few organisms are in stages of division. Multifocally, there is mineralization of necrotic areas. As was visible grossly, a green hue of some organisms can be seen in the paraffin sections, not clearly visible in the methacrylate sections. The organisms were identified as green algae by their color and morphology, consistent with descriptions of Chlorella. Electron microscopy of formalin-fixed material revealed chloroplasts within unidentified developmental stages of the algae.
Chlorella is a chlorophyll-containing green alga that seems to be a rare pathogen in mammals. It must be differentiated from Prototheca species only. Unlike Prototheca, the organisms contain abundant cytoplasmic, PAS-positive granules and chloroplasts. Chloroplasts can be proven by electron microscopy. Chlorellosis is described in cattle, humans, a lamb, and (most likely) a beaver. All tissues affected had the characteristic bright green color in common. We could not find a more recent report in the literature.
AFIP Diagnosis: Stratified squamous epithelium overlying fibrous connective tissue (skin, per contributor): Inflammation, granulomatous, diffuse, severe, with granulation tissue and numerous endosporulating algae, gazelle (Gazella dorcas), artiodactyla.
Conference Comment: Electron micrographs submitted by the contributor reveal a 12 m m diameter, irregularly round algal organism surrounded by collagen fibers and electron lucent granular space. The algal organism has a 0.5 m m thick cell wall with the outer half of the wall being composed of an electron dense granular layer and the inner half being more lucent and composed of two layers of fibers. The outer half of these fibers is arranged perpendicularly to a dense granular layer. The more central layer of fibers is oriented circumferentially.
The alga contains numerous electron dense, round granules (starch) and numerous chloroplasts consisting of membranes stacked in bands of 4 to 6 lamellae. The chloroplasts frequently form a ring with a center of fibrils that radiate from a more dense, granular center. No nucleus or other identifiable organelles are apparent.
Differentiating Chlorella from other endosporulating organisms such as Prototheca spp., Rhinosporidium spp.and Coccidioides immitis is not difficult. Grossly, a granulomatous cellulitis with green color must be differentiated from eosinophilic inflammation. Histologically, the presence of endosporulating spheroidal organisms with an average diameter of 8-11 m m, and many large, strongly PAS-positive granules in the cytoplasm are supportive of chlorellosis.
Contributor: University of Munich, Institute of Veterinary Pathology, Veterinaerstrasse 13, D-80539 Munich, Germany
References: 1. Chandler FW, Kaplan W, Callaway CS: Differentiation between Prototheca and morphologically similar green algae in tissue. Arch Pathol Lab Med
102:353-356, 1978
2. Cordy DR: Chlorellosis in a lamb. Vet Pathol 10:171-176, 1973
3. Gibbs SP: The comparative ultrastructure of the algal chloroplast. Annals NY Acad Sci 175:454-473, 1970
4. Modly CE, Burnett JW: Cutaneous algal infections: Protothecosis and Chlorellosis. Cutis 44:23-24, 1989
5. Nadakavukaren MJ, McCracken DA: An ultrastructural survey of the genus
Prototheca with special reference to plastids. Mycopathologia 61:117-119, 1977
6. Rogers RJ, Connole MD, Norton J, Thomas A, Ladds PW, Dickson J: Lymphadenitis of cattle due to infection with green algae. J Comp Path 90:1-9, 1980
7. Sileo L, Palmer NC: Probable cutaneous protothecosis in a beaver. J Wildlife Diseases 9:320-322, 1973
CASE III – Rat A or B (AFIP 2740423)
Signalment: Male Fischer 344 rats, 110-120 g
History: Rats were dosed with a developmental pharmaceutical compound or the carrier vehicle by gavage for 14 days. Rat A is a control rat. Rat B was given 100 mg compound/kg body weight.
Gross Pathology: At necropsy, livers from treated rats were enlarged and pale. Some affected livers had few to multiple, small (pinpoint) white and/or red lesions. Absolute and relative liver weights were double that of control rats.
Laboratory Results: Clinical pathology parameters were collected on non-fasted rats on Day 3, and on fasted rats on Day 14. On Day 14, in comparison to controls, the treated rats had increased ALT (360%), AST (218%), ALP (46%), triglycerides (200%), cholesterol (111%), albumin (17%), and A/G ratio (132%), with decreased total protein (1.3%) and globulins (49%). With the exception of cholesterol and triglycerides, alterations in the other parameters on Day 3 (non-fasted samples) were similar, but of lesser magnitude. On Day 3, serum triglycerides and cholesterol were decreased (83% and 29%, respectively) in comparison to controls.
Histologically, treated rats had diffuse hepatocellular hypertrophy characterized by granular eosinophilic cytoplasm and loss of cytoplasmic basophilia. Increased mitoses, increased numbers of binucleate hepatocytes, and karyomegaly (compared to controls) were noted in some treated rats. Foci of coagulative hepatic necrosis with hemorrhage and/or subacute to chronic inflammation correspond to red or white foci, respectively, noted in some livers at necropsy.
Ultrastructurally, in comparison to liver from control rats (Rat A), hepatocytes from treated rats (Rat B) are enlarged (hypertrophy) approximately 2-3 times, and have marked increases in the number and size of peroxisomes. Peroxisomes are variably sized (approximately 0.5-3 m m), sometimes elongated, and fill the cytoplasm, displacing other cellular organelles. Profiles of smooth endoplasmic reticulum (minimal increases compared to control Rat A), rough endoplasmic reticulum and mitochondria are interspersed between the peroxisomes. Multifocally, the endoplasmic reticulum is dilated and contains a scant amount of osmophilic (proteinaceous) material. Hepatocytes in the treated rat have low numbers of small lipid droplets and lysosomes. Both electron micrographs are centered on centrilobular (periacinar) hepatocytes. A central vein is present in both micrographs.
Contributor’s Diagnosis and Comment: 1. Rat A: Normal liver.
2. Rat B: Hepatocellular hypertrophy with marked peroxisomal hypertrophy and proliferation and minimal proliferation of SER.
Liver normally contains an abundant number of peroxisomes. Peroxisomes range in size from 0.1-1.5 m m in diameter, are surrounded by a thin, single-membrane, have a fine granular matrix and, in some species such as the rat, a dense central core (nucleoid). Hypolipemic drugs, such as clofibrate, are well known to cause peroxisomal proliferation via activation of peroxisome proliferator-activated receptors (PPAR), primarily PPARa , and to a much lesser extent, PPARg . The developmental compound administered in this example is believed to cause peroxisomal proliferation through a similar mechanism.
Hepatocarcinogenesis in rodents is associated with chronic stimulation of PPARa by non-genotoxic peroxisome proliferators. Increases in serum hepatic enzymes correlate to hepatocellular hypertrophy and necrosis and enlarged livers. Such changes are consistent with reported hepatic pre-neoplastic lesions in rodents.
PPAR are involved in lipid and glucose metabolism, cell differentiation, cell cycle withdrawal, and inflammation. The clinical pathology data here is consistent with the expected response in comparison of fed to fasted rats with stimulation of PPAR activity.
AFIP Diagnosis: Liver: Hepatocellular hypertrophy, with marked peroxisomal proliferation and hypertrophy, Fischer 344 rat, rodent.
Conference Comment: In the electron micrograph from control rat A there are at least 3 cuboidal cells arranged around a blood vessel (central vein) which contains an erythrocyte. The cells have irregular, blunt apical microvillar projections that extend to the endothelial cells lining the central blood vessel (space of Dissé). The cells have apical tight junctions and numerous desmosomes. Approximately mid-way along the cell lateral border, desmosomes isolate small canaliculi that have microvillar projections into the lumen (bile canaliculi). The cytoplasm is filled with abundant smooth endoplasmic reticulum, lesser amounts of rough endoplasmic reticulum, oval to elongate mitochondria, lysosomes, and one cell contains a large fat droplet. Each cell has a central, round nucleus with evenly dispersed euchromatin centrally and a thin band of heterochromatin peripherally. The contributor has described the ultrastructural changes in the tissue from rat B.
Peroxisomes are membrane-bound cytoplasmic organelles containing enzymes involved in beta-oxidation of fatty acids, the biosynthesis of cholesterol, and other biochemical pathways. PPARa plays a role in regulation of fatty acid beta-oxidation, apolipoproteins, and fatty acid transport proteins.
Peroxisome proliferators are a diverse group of chemicals that include hypolipidemic drugs, plasticizers and organic solvents, herbicides, and naturally occurring hormones. They cause an increase in the number and size of peroxisomes in the liver, kidney, and heart of susceptible species, such as rats and mice, and can cause hepatocellular neoplasia in these animals with long-term administration through mechanisms that do not involve direct DNA damage. Long-term administration of hypolipidemic drugs has not been shown to increase the risk of cancer in humans; however, it is unknown whether frequent exposure to peroxisome proliferators represents a hazard to humans. The hypolipidemic drugs are effective at reducing free cholesterol that escapes hepatic scavenging, but the mechanisms by which humans and nonhuman primates are resistant to peroxisome proliferation are not known.
Contributor: Eli Lilly and Company Research Laboratories, P. O. Box 708, 2001 W. Main Street, Greenfield, IN 46140
References: 1. Excher P, Wahli W: Peroxisome proliferator-activated receptors: insight into multiple cellular functions. Mutation Res 448:121-138, 2000
2. Gonzalez FJ, Peters JM, Cattley RC: Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activated receptor a . J Nat Cancer Inst 90:1702-1709, 1998
CASE IV – 00-1142 (AFIP 2737283)
Signalment: 11-year-old, spayed female, Great Dane
History: The dog had a rapidly growing, dark pedunculated mass attached to the inner surface of the left lower lip.
Gross Pathology: The globoid mass was 1.5 x 1.5 x 2.5 cm, grey-white, and had an attached small stalk.
Laboratory Results: None.
Contributor’s Diagnosis and Comment: Oral mucosa of lip: Osteoma and invasive melanoma.
An undecalcified section contains a pedunculated mixed tumor comprised primarily of osteoma and invasive melanoma. Spindle-shaped and epithelioid melanocytes, with varying amounts of melanin, bridge the base of the pedicle (seen as an epithelial-covered tag in some sections). There is extensive junctional activity, both in the pedicle and scattered around the circumference. Within the submucosa of the pedicle, there are variable numbers of melanophages. Some sections have invasive melanoma. Some venules have what appears to be melanin within the endothelium, but the birefringent black pigment is acid hematin.
Beneath the partially ulcerated mucosa, there is a fibrous tumor, with multifocal nests of epithelioid melanocytes. The fibrous component quickly merges with osteoblasts and chondroblasts, with endochondral bone (in the region opposite the pedicle). These cells give rise to the large, central, partially mineralized osteoma. The osteoma component is composed of numerous branching trabeculae with broad, fibrous osteoid seams of woven bone, often lined by a pavement of osteoblasts. Segments of these seams are devoid of osteocytes, but lacunae with several osteocytes are common. Osteoclasts are commonly applied to the bony surfaces and some are in Howship’s lacunae. There is limited remodeling to lamellar bone. Occasionally, smaller satellite foci of osteoid arising in the fibrous component may be seen in some sections.
This tumor is only superficially similar to the canine oral osteoid melanoma recently reported by Chénier and Doré. That first report of osteoid formation within a malignant melanoma describes melanocytes intimately associated with osteoid. The most likely explanation for this association is melanocytes secreting a cytokine, such as a transforming growth factor, which transforms fibroblasts to osteoblasts. There is no evidence that melanocytes can directly produce osteoid, but collagen formation in melanoma cells has been documented by electron microscopy.
In this pedunculated tumor, there are 2 distinct components of osteoma and melanoma. No melanocytes are intimately associated with the osteoid deposition. It is conceivable melanocytes induced the osteoma. The diagnosis of osteoma is based on the formation of regular trabeculae, atypical acellular osteoid seams, and multiple cells within osteocyte lacunae.
There is no recurrence of the tumor 2.5 months after its excision.
AFIP Diagnosis: Oral mucosa: Malignant melanoma, with reactive bone, Great Dane, canine.
Conference Comment: This case was reviewed in consultation with the AFIP's Department of Orthopedic Pathology. Our interpretation of the central bony tissue is that it represents a reactive, rather than neoplastic, process. A peripheral, highly cellular, periosteum-like zone surrounds well-formed cartilage and bony trabeculae. The trabeculae are lined by numerous osteoblasts in more peripheral areas.
The presence of mesenchymal tissues such as bone, cartilage, or adipose tissue in sites that do not normally contain these elements is classified as connective tissue metaplasia. In a metaplastic change, alterations in signals generated by cytokines, growth factors, and components of the extracellular matrix result in the reprogramming of stem cells or of undifferentiated mesenchymal cells present in connective tissue, causing them to differentiate along a new pathway. Members of the transforming growth factor-b superfamily are known to induce chondrogenic or osteogenic expression in mesenchymal stem cells. The exact pathogenic cascade that ultimately results in tissue metaplasia is unclear in most instances.
Osteomas are solitary, benign, bony growths protruding from the surface of bone, usually on the head, that blend with underlying resident bone and are composed of essentially normal, mature, well-differentiated bone that is typically formed by intramembranous ossification.
Contributor: University of Tennessee, College of Veterinary Medicine, Department of Veterinary Pathology, Room A201, Knoxville, TN 37901-1071
Reference: 1. Chénier S, Doré M: Oral malignant melanoma with osteoid formation in a dog. Vet Pathol 36:74-76, 1999
2. Cotran RS, Kumar V, Collins T: Cellular pathology II: adaptations, intracellular accumulations, and cell aging. In: Robbins Pathologic Basis of Disease, 6th ed., pp. 36-38. WB Saunders Co, Philadelphia, PA, 1999
3. Palmer N: Bones and joints. In: Pathology of Domestic Animals, ed. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 1, pp. 4-16. Academic Press, San Diego, CA, 1993
4. Pool RR: Tumors of bone and cartilage. In: Tumors
in Domestic Animals, ed. Moulton JE, 3rd ed., pp. 159-162.
University of California Press, Berkeley, CA, 1990
*Sponsored by the American Veterinary Medical Association, the American College of Veterinary Pathologists and the C. L. Davis Foundation.
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