AFIP Wednesday Slide Conference - No. 19

February 3, 1999
Conference Moderator:
Dr. Michael A. Eckhaus, Diplomate, ACVP
NCRR LSS SSB, Bldg. 28A, Room 117
28 Library Drive, MSC 5210
Bethesda, MD 20892-5210
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Case I - OL8115 (AFIP 2648175)

Signalment: Ten-week-old, Brown Norway rats.
History: The rats were quality-control animals. They showed no clinical signs.
Gross Pathology: There were multiple, small, brown to red foci over the pleural surface of all lung lobes

Laboratory Results:
1. Serology: Negative.
2. Parasitology: Negative.
3. Microbiology: Staphylococcus aureus, Staphylococcus xylosus, Streptococcus sanguis, and Streptococcus bovis were recovered from the nasopharynx.
Contributor's Diagnosis and Comments: Lung: Peribronchiolar and interstitial pneumonia, granulomatous and eosinophilic, multifocal (varied severity among sections).

Etiology: Unknown.

Multifocally throughout the lung there are variably-sized, discrete, noncaseating granulomas, mostly arranged around bronchioles, but also present around or adjacent to vessels in the interstitium. Granulomas are usually composed of tightly packed epithelioid macrophages admixed with frequent multinucleated, Langhans-type and foreign body-type giant cells and mild to moderate numbers of eosinophils and neutrophils. Within granulomas, there are occasional small central areas of necrosis characterized by accumulation of cellular and nuclear debris, degenerate polymorphonuclear leukocytes, and rarely, deposition of small amounts of brightly eosinophilic, club-shaped, amorphous material (Splendore-Hoeppli material; not present in all sections). In addition, throughout the interstitium there are small to conspicuous perivascular cuffs composed mostly of eosinophils and neutrophils, admixed with occasional lymphocytes. In association with inflammatory infiltrates, there is also mild to moderate alveolar histiocytosis, mild to moderate interstitial edema, mild thickening of alveolar walls with type II pneumocyte hyperplasia, and mild to moderate hyperplasia of bronchiolar epithelia with mucous metaplasia.
Multifocal granulomatous pneumonia is a condition affecting Brown Norway rats. This condition has been observed worldwide, but its exact incidence is not known. The only published information reported this condition in 11 of 12, 10-week-old, Brown Norway rats, and in 7 of 10 retired breeders (1). Based on macroscopic examination, the condition can be diagnosed in approximately 25% of 8 to 10-week-old Brown Norway rats. Females appear more frequently affected than males, and the incidence in retired breeders is lower, suggesting that this condition may regress (Charles Clifford, Charles River Laboratories, personal communication). The pulmonary lesions are not associated with clinical signs and do not affect the lifespan of the rats.
Grossly, multiple gray to gray-brown foci can be observed over the pleural surface of all pulmonary lobes. Histologically, the condition is characterized by multifocal dense aggregates of mostly macrophages admixed with frequent multinucleated giant cells, eosinophils, and neutrophils, with fewer lymphoid cells. The inflammatory infiltrates are peribronchiolar and interstitial, but usually not in airway lumens.
The cause of this condition is unknown. Serology, microbiologic tests and special stains (Warthin-Starry, acid-fast, Gram) do not demonstrate an infectious agent. In addition, the condition does not appear to be contagious, as rats of other strains housed in the same rooms do not develop the pulmonary lesions. Recently, pulmonary inflammatory lesions of unknown etiology have been reported in Fischer 344 rats used in chronic toxicity studies (2). These lesions were similar to those of Brown Norway rats in that they were more commonly observed in younger animals. However, the inflammatory infiltrates in Fischer 344 animals were mostly lymphocytic, in contrast to the histiocytic infiltrates of Brown Norway rats suggesting that these two conditions are different. Alternatively, the different nature of the inflammatory infiltrates may simply reflect strain differences in inflammatory response (3,6).
In humans, multiple types of noninfectious granulomatous pulmonary conditions of unknown etiology are recognized, including sarcoidosis, Wegener's granulomatosis, Histiocytosis X (pulmonary Langerhans cell granulomatosis), and hypersensitivity pneumonitis (4,5,7). There is some overlap in the type, distribution, and histologic appearance of pulmonary lesions among these disorders. The exact cause of these conditions is unknown, but is most likely related to an uncontrolled response of the immune system (4,5,7).
Brown Norway rats have been used as models of allergic respiratory disease such as asthma, because of their high capacity for IgE production and their airway hyperresponsiveness following exposure to allergens (such as ovalbumin) or some chemicals (1). In addition, this strain is known for the development of autoimmune syndromes following administration of mercuric chloride, gold, and penicillamine (6). Compared to other strains of rats, Brown Norway rats have low numbers of CD8+ T cells, CD8+ CD45RChigh, and CD4+ CD45RChigh (6). Because of their unique immune system and the histologic nature of the lesions, it is tempting to speculate that this granulomatous pneumonia may result from a disordered immune response to an unknown antigen. Recently, small, noncaseating granulomas containing giant cells have been observed in the lungs of Brown Norway rats following mercuric chloride administration (6). The lesions described were very similar to this idiopathic granulomatous pneumonia of Brown Norway rats. This finding suggests that this common background lesion may represent a confounding factor for the interpretation of studies involving Brown Norway rats.
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Case 19-1. Lung. Multifocally throughout the lung, abundant macrophages, eosinophils, neutrophils and scattered foreign body or Langhans giant cells expand or replace alveoli and bronchioles.
AFIP Diagnosis: Lung: Pneumonia, granulomatous and eosinophilic, peribronchiolar and perivascular, multifocal, moderate, with perivascular edema, Brown Norway rat, rodent.
Conference Note: Like the contributor, participants identified perivascular and peribronchiolar inflammatory infiltrates composed of macrophages with multinucleate giant cells and eosinophils, and fewer lymphocytes and plasma cells. Inflammatory cells expand adjacent alveolar septa, and sometimes obscure or fill alveoli, but are rarely found within bronchiolar airways. Various histochemical stains performed at the AFIP (tissue Gram stains, acid-fast stains, periodic-acid Schiff reaction, and Grocott's methenamine silver method) did not demonstrate the presence of an infectious agent.
The contributor noted the recent report of perivascular inflammatory lesions in the lungs of Fischer 344 rats composed predominately of lymphocytes with an associated alveolar exudate of macrophages, neutrophils, and lymphocytes. An increase in peribronchiolar lymphoid tissue was also observed. More recently, pulmonary inflammatory lesions with histomorphologic features similar to those in F344 rats were described in Wistar rats.
While still of unknown etiology, several common features suggest that these strain-specific lesions may have a similar pathogenesis: lack of clinical signs in affected animals, distribution of inflammatory infiltrates, presence of more extensive inflammatory lesions in young animals, and age-related spontaneous regression of lesions. While the character of the inflammation in the Brown Norway rat is different from the other two strains, i.e. granulomatous and eosinophilic versus lymphocytic, this may be attributed to variation in immune response as suggested by the contributor.
The etiology of the inflammatory lesions was not determined in the Brown Norway or Wistar rats. In the report of F344 rats, variable (often small) numbers of rod-shaped bacteria were observed ultrastructurally within macrophages or degenerate cells in alveoli of most animals with lung lesions.
Contributor: Searle, 4901 Searle Parkway, Skokie, IL 60077.
1. Ohtsuka R, Doi K, Itagaki S: Histological characteristics of respiratory system in Brown Norway rat. Exp Anim 46:127-133, 1997.
2. Elwell MR, Mahler JF, Rao GN: Inflammatory lesions in the lungs of rats. Toxicol Pathol 25:529-531, 1997.
3. Sorden SD and Castleman WL: Brown Norway rats are high responders to bronchiolitis, pneumonia, and bronchiolar mastocytosis induced by parainfluenza virus. Exp Lung Res 17:1025-1045, 1991.
4. Jones WW, Geraint JD: Pulmonary Langerhans' cell granulomatosis (LCG). Sarcoidosis 10:104-107, 1993.
5. Soler P, Tazi A, Hance AJ: Pulmonary Langerhans cell granulomatosis. Cur Opi Pulmon Med 1:406-416, 1995.
6. Qasim FJ, Thiru S, Mathieson PW, Oliveira DB: The time course and characterization of mercuric chloride-induced immunopathology in the Brown Norway rat. J Autoimmun 8:193-208, 1995.
7. Fleming MV, Travis WD: Interstitial lung disease. Pathol 4:121, 1996.
8. Slaoui M, Dreef C, van Esch E: Inflammatory lesions in the lungs of Wistar rats. Toxicol Pathol 26:712-713, 1998.

Case II - 1971508 (AFIP 2642673)

Signalment: Seven-week-old, Jack Russell terriers, canine.
History: Seven 7-week-old puppies were examined because of a history of unexplained mortality in previous litters. Six of 14 puppies from three litters of the same breeding pair died unexpectedly at eight weeks of age shortly after vaccination. Administration of modified live virus vaccines produced hepatic necrosis with inclusions, characteristic of adenoviral hepatitis. In this case, four unvaccinated puppies from a litter of seven were chosen based on the finding of lymphopenia. There were no significant clinical signs in any of the animals. These pups were examined at necropsy, along with two non-lymphopenic littermates.
Gross Pathology: In four of the six puppies necropsied, the thymus was markedly hypoplastic in appearance and weighed from 0.5 to 2 grams. In contrast, the two non-lymphopenic litter mates had thymic weights of 8.5 and 10 grams. Hypoplasia of lymph nodes and hyposplenism was also noted in the four lymphopenic puppies. Other gross lesions in the lymphopenic pups included diffuse, mild catarrhal enterocolitis and, in one pup, mild, acute multifocal necrotizing hepatitis. All six animals were severely flea infested, but had no internal parasites.
Laboratory Results: The four animals with reduced thymic weight had leukopenia with severe relative and absolute lymphopenia (an average of 2% lymphocytes ranging from 0-5% of the total WBC with an absolute count of 0.1 x 103/ml ranging from 0-0.2 x 103/ml), a mild relative neutrophilia, and severely reduced IgM (undetected with all values less than 10 mg/100 ml in the four lymphopenic animals compared with non-lymphopenic litter mates having a mean of 173 mg/100 ml ranging from 140-200 mg/100 ml) and an IgG (mean of 115 mg/100 ml ranging from 60-200 mg/100 ml compared with non-lymphopenic litter mates having a mean of 417 mg/100 ml ranging from 350-500 mg/100 ml). All animals had a moderate regenerative anemia and mild monocytosis. Platelet counts were normal. Previous litters had no blood count data, but a fourteen-week-old, unvaccinated pup had undetectable IgA, IgG and IgM a few days before death.
Contributor's Diagnoses and Comments:
1. Marked thymic cortical hypoplasia.
2. Severe combined immunodeficiency (SCID), Jack Russell terrier.
The thymic tissues presented for examination represent age and sex-matched controls of affected versus unaffected littermates. Affected puppies had markedly diminished thymic mass, with primary depletion of thymic cortical elements. Lymphoid hypoplasia was also present (tissues not submitted) in the spleen (no periarteriolar sheaths or follicles), lymph nodes, and GALT/BALT. The immunophenotypic analysis of lymphocytes by cluster of differentiation (CD) typing is not yet available. The remarkable paucity of circulating lymphocytes did not afford the opportunity for in vitro mitogen stimulation.
The marked lymphoid hypoplasia and insignificant amounts of IgM in seven-week-old animals, coupled with a history of death at the time of maternal antibody titer loss, strongly suggests a novel, severe combined immunodeficiency (SCID) of Jack Russell terriers. Further, the one to one female to male ratio among 10 affected animals of 24 at risk with the same, clinically normal sire and dam suggests an autosomal recessive trait. Acquired immunodeficiencies occur in the canine, but are usually characterized by incomplete lymphopenia, deficiencies of single or multiple components of the immune system, and often a marked proliferative response in phagocytic (reticuloendothelial) systems.
Canine severe combined immunodeficiency has been reported in the Basset Hound and Cardigan Welsh Corgi breeds to date. In these breeds, the disease is inherited as an X-linked recessive trait (XSCID), and thus affects only male puppies. The disease has been determined to be due to a four-nucleotide deletion defect in the genetic locus coding for the gamma chain of the interleukin-2 receptor (IL-2R).1 Affected puppies have increased proportions of immature thymocytes, normal relative numbers of B-lymphocytes, and normal IgM levels, although T-lymphocytes (particularly the CD8+ subset), IgG, and IgA percentages tend to be reduced or are absent.
In the human being, forms of severe combined immunodeficiency include an adenosine deaminase deficiency, a defect in RAG-1 or RAG-2, and reticular dysgenesis (may be X-linked inheritance with deficient T-cells and normal or high levels of B-cells, or autosomal recessive inheritance with T-cells and B-cells both deficient).3,4

The most common form of human SCID is also X-linked recessive in inheritance, and is similar to the disease previously reported in canines in that it involves a defect in the IL-2R gamma chain locus. The second most prevalent form of human SCID involves an autosomal recessive inheritance pattern-based defect in production of the purine nucleoside metabolizing enzyme, adenosine deaminase (ADA).4 Defects in the production of the purine metabolizing enzyme purine nucleoside phosphorylase (PNP) and defects in expression of MHC class II account for some of the remaining heritable human SCID cases, though these conditions are much more rare than are human XSCID and ADA deficiencies.4,5
The disease present in this litter of Jack Russell terriers represents the first description of non-X-linked SCID in a canine breed. Both male and female puppies in this litter were affected, as evidenced by absence of circulating lymphocytes and lymphoid hypoplasia in multiple tissues. Additional studies to determine whether this litter may have deficiencies in one or more purine catabolizing enzymes such as ADA, similar to the more common non-X-linked human SCID variants, are being performed at the time of this submission. Equine SCID, which is most common in the Arabian breed, is inherited as an autosomal recessive trait. The defect in equine SCID is an absence of the p350 component of DNA-dependent protein kinase.6 Such a defect in these dogs cannot be excluded at this time.
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Case 19-2. Thymus. There is diffuse, marked depletion of lymphocytes and no distinction between cortical and medullary zones. Reticular stromal cells remain with few small lymphocytes.
AFIP Diagnoses:
1. Thymus and lymph node: Hypoplasia, lymphoid, diffuse, severe, Jack Russell terrier, canine.
2. Thymus and lymph node: Extramedullary hematopoiesis, diffuse, mild to moderate.
Conference Note: Due to section variation, some slides may contain only lymph node and no thymus. In the affected thymus, there is marked decrease in size of thymic lobules, loss of demarcation between the cortex and medulla, and prominence of medullary epithelial reticular cells due to marked depletion of lymphocytes. In lymph node sections, there is marked depletion of lymphocytes, with virtual absence of paracortical lymphoid cells. Immunohistochemical staining for CD3, a pantropic T-lymphocyte marker, performed at the AFIP demonstrated diffuse marked decrease of CD3-positive cells as compared with the thymus of the age-matched control littermate, especially within the medulla where mature T-cells are found. Small numbers of CD3-positive T-cells are present in the cortex (immature T-lymphocytes).
In horses, SCID is inherited as an autosomal recessive disorder in Arabian and Appaloosa breeds. Affected foals lack cell-mediated immunity and are unable to synthesize immunoglobulins. Foals may remain normal during the first few months of life owing to colostral transfer of maternally derived IgG, but as passive immunity wanes, the foals become increasingly susceptible to infections. The disorder is characterized by lymphopenia, decrease or absence of immunoglobulins, and death by five months of age. Death most commonly results from pneumonia caused by adenovirus, Rhodococcus equi, Pneumocystis carinii, or Cryptosporidium parvum. The thymus is aplastic or hypoplastic, and the spleen and lymph nodes lack follicles and contain few lymphocytes and no plasma cells.

Contributor: Animal Health Diagnostic Laboratory, P.O. Box 30076, Lansing, MI 48909.
1. Pullen RP, Somberg RL, Felsburg PJ, Henthorn PS: X-linked combined immunodeficiency in a family of Cardigan Welsh Corgis. J Amer Anim Hosp Assoc 33:494-499, 1997.
2. Felsburg PJ, Somberg RL, Perryman LE: Domestic animal models of severe combined immunodeficiency: Canine X-linked severe combined immunodeficiency and severe combined immunodeficiency in horses. Immunodeficiency Reviews 3:277-303, 1992.
3. Blaese RM: Genetic immunodeficiency syndromes with defects in both T and B-lymphocyte function. In: The Metabolic and Molecular Bases of Inherited Disease, Scriver CR, Beaudet AL, Sly WS, Valle D, eds., vol. 3, pp. 3895-3909, McGraw-Hill, New York, 1995.
4. Hershfield MS, Mitchell BS: Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. In: The Metabolic and Molecular Bases of Inherited Disease, Scriver CR, Beaudet AL, Sly WS, Valle D, eds., vol. 3, pp. 1725-1768, McGraw-Hill, New York, 1995.
5. Rosen FS, Bhan AK: A 54-day-old premature girl with respiratory distress and persistent pulmonary infiltrates. New Engl J Med Surg 338:1752-1759, 1998.
6. Tizard IR: Primary immune deficiencies. In: Veterinary Immunology: An Introduction, 5th edition, pp. 445-447, W.B. Saunders, Philadelphia, PA, 1996.
7. Jones TC, Hunt RD, King NW: Immunopathology. In: Veterinary Pathology, 6th ed., pp. 186-189, Williams and Wilkins, Baltimore, MD, 1997.

Case III - 98-7050 or 98-7442 (AFIP 2641860)

1. 98-7050: 2-month-old, female BALB/cJ-Fechtm1Pas/Fechtm1Pas mouse.
2. 98-7442: 5-month-old, female BALB/cJ-Fechtm1Pas/Fechtm1Pas mouse.
Both animals are inbred laboratory mice. The scientific name is inappropriate, since inbred mice are derived from a variety of substrains.
History: Both mice were submitted for a limited study. They were placed under lights in a mouse room to determine if they routinely devel-oped photosensitization problems. This did not happen in a conventional mouse room. Ultraviolet lights of specific wavelengths are required to induce skin lesions. This was done to address an animal health concern raised by a clinician.
Gross Pathology: Mice submitted were alert and active. The livers of both mice were firm and dark brown to purple, with irregular and roughened surfaces. In case 98-7050, the urine was observed to be orange.

Laboratory Results: None.
Contributor's Diagnoses and Comments:
1. Liver, mild bile duct hyperplasia.
2. Liver, moderate biliary fibrosis.
3. Liver, mild chronic periportal hepatitis.
4. Liver, porphyrin cholelithiasis.

Etiology: Autosomal recessive mutation, ferrochelatase deficiency.
Contributor's comments consist of text taken directly from: Montagutelli X: The ferrochelatase deficiency (Fechm1Pas) mutation, chromosome 18. In: Handbook of Mouse Mutations with Skin and Hair Abnormalities, Sundberg JP, ed., pp. 247-251, CRC Press Inc., Boca Raton, FL, 1994.
The ferrochelatase deficiency (Fechm1Pas) mutation arose in 1988 at the Institut Pasteur in Paris in a mutagenesis experiment with ethylnitrosourea.1 The first features observed were an intense yellow color of the serum, reduced hematocrit, and grossly evident jaundice in albino mutant mice. This condition was transmitted in an autosomal recessive manner. The mutation was introduced, through several backcrosses, onto the BALB/cByJ inbred background, where the anomalies appeared to be the most severe.
The influence of genetic background on the development of jaundice has been observed through the intercrossing of heterozygotes derived from the cross of homozygous mutants (close to BALB/cByJ) with (-C57BL/6J X SJL/J)F1 hybrids. This intercross did not yield affected mice with overt jaundice, even though other biological parameters, such as elevation of protoporphyrin (see below) were characteristic for this mutation (Montagutelli, unpublished data). The ferrochelatase deficiency mutation was mapped to mouse chromosome 18, 40 cM from the centromere (Montagutelli, unpublished).
Gross Lesions: On the BALB/cByJ background, mutant mice can be recognized as early as two weeks of age by the intense yellow color of their serum and by gross bilirubinuria. Jaundice is apparent by the yellow coloration of the unpigmented ears. Photosensitivity is often observed in homozygotes under standard husbandry conditions (fluorescent lighting). Inflammatory lesions appear primarily on the ears, which initially become red and swollen. In some mice, ear tips undergo necrosis, and multiple ulcerations develop. Necrotic ear tips undergo autoamputation which eventually heal, leaving deformed pinnae. Adult homozygotes have an enlarged abdomen due to marked hepatomegaly and splenomegaly, which is progressive from the first days of life. On the BALB/cByJ background, males are usually fertile whereas females breed rarely. No anomalies have been observed in heterozygotes, other than a transient and mild jaundice that may be seen at 4 to 5 weeks of age.
Microscopic Lesions: Very severe liver lesions are observed in homozygous ferrochelatase deficiency mutant mice. At 15 days of age there is a 65% increase in the liver:total body weight ratio. Microscopic examination reveals portal and periportal fibrosis (Fig. 2) as well as focal accumulation of dense, dark brown pigment in canaliculi, interlobular biliary ducts and Kupffer cells (Fig. 3). Erythroid hyperplasia is prominent in the bone marrow and spleen.
A normocytic anemia develops after one month of age in homozygotes. At six months of age, hemoglobin concentration, red blood cell counts, and hematocrits are decreased by 25-30% that of controls or heterozygotes. Red blood cells are more heterogeneous, as determined by the increased volume range distribution width and the heterogeneity of cell resistance to osmotic lysis. Polychromasia, anisocytosis, target cells, and leptocytes are observed in blood films.
Immunological and Biochemical Abnormalities: Plasma bilirubin (mainly conjugated bilirubin) is markedly increased (50 fold) in homozygotes. Protoporphyrin levels are also considerably elevated in erythrocytes (25 fold), plasma (20-200 fold), liver (1000 fold) and stool (10 fold). Serum alkaline phosphatase and transaminases are consistently increased, as a consequence of chronic liver damage. Enzymatic activity of ferrochelatase in spleen, kidney, and liver in homozygotes is 3-7% of normal controls and close to 50% of normal in heterozygotes.
Ferrochelatase is the last enzyme of the heme biosynthesis pathway that catalyzes the insertion of ferrous iron (Fe2+) into protoporphyrin.2 The cDNA that encodes for ferrochelatase has been sequenced.3 In the ferrochelatase deficiency mouse mutation, a T to A transposition at nucleotide 293 was identified in the gene coding for the defective enzyme. This transposition led to a methionine to lysine substitution at position 98 in the protein M98K.4 In vitro expression of the mutant protein leads to reduced enzymatic activity, similar to that observed in vivo.
Analogous Human Disease: In humans, erythropoietic protoporphyria (EPP) is associated with reduced activity of ferrochelatase.2,5 The disease is characterized by cutaneous photosensitivity. A mild microcytic, hypochromic anemia is observed in a minority of cases. Fatalities from rapidly progressive liver disease have been reported in at least 20 patients,2,6 which is an indication for liver transplantation.7-10 Biochemically, EPP results in the accumulation of protoporphyrin in erythrocytes, plasma, and feces. EPP is generally assumed to be an autosomal dominant hereditary condition,2,11 but it may be inherited, in some cases, in an autosomal recessive fashion.12-14 Four human mutations have been described so far.
Analogous Animal Diseases: Ferrochelatase deficiency has been described in cattle.18 Affected cattle develop cutaneous lesions after exposure to sunlight but they do not develop anemia or hepatobiliary disease.19,20 Bovine protoporphyria is transmitted as an autosomal recessive trait.
Potential Uses of the Ferrochelatase Deficiency Mutation: The ferrochelatase deficiency mutation is the first murine genetically determined model for human EPP. Because of the high incidence and severity of liver disease in the mouse, which represents the main complication in the human, this model is likely to become highly used to investigate the human disease and test new therapies.

Availability of Mice: Mice are currently available as heterozygous breeding pairs on a limited basis from investigators at the Institut Pasteur in Paris, France. This mutation has been imported into the Induced Mutant Resource at The Jackson Laboratory, Bar Harbor, Maine, USA where it is readily available as a second resource.
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Case 19-3. Liver. There is diffuse bile duct hyperplasia. These bile ducts are often surrounded by lymphocytes and plasma cells. Scattered foci of neutrophils and intrahepatocytic brown pigment are also present.
AFIP Diagnosis: Liver: Hyperplasia, biliary and oval cell, portal and periportal, diffuse, moderate, with multifocal mild lymphocytic and neutrophilic portal and periportal hepatitis, individual hepatocyte necrosis, and intracellular brown globular anisotropic pigment, BALB/cJ-Fech mouse, rodent.
Conference Note: Porphyrias are uncommon disorders caused by disturbances or deficiencies of enzymes involved porphyrin metabolism. Porphyrins are pigments found in hemoglobin, myoglobin, and cytochromes. Enzyme deficiencies of heme biosynthesis lead to excessive accumulation of porphyrins and their precursors. In erythropoietic porphyrias, enzyme deficiencies may occur during the formation of protoporphyrin, or, as in this case, at the last step of heme synthesis. The intermediates that accumulate during dysfunctional heme synthesis, and the resultant clinical manifestations, depend upon the step at which the enzymatic defect occurs.
Congenital erythropoietic porphyrias have been documented in several domestic animal species, including several breeds of cattle, swine, and in domestic shorthair and Siamese cats. The disorder is inherited as an autosomal dominant trait in pigs and cats, while in cattle it is an autosomal recessive disorder. Porphyrias resulting from an enzymatic defect of uroporphyrinogen III cosynthetase, such as in cattle and humans, cause overproduction of uroporphyrin I, coproporphyrin I, and protoporphyrin III, which escape the erythrocyte and accumulate in tissues. In cattle, accumulation of these pigments in bone and dentine leads to pink-red discoloration, known clinically as osteohemochromatosis and "pink-tooth", respectively. Anemia occurs due to abnormal hemoglobin production and decreased erythrocyte life span. Increased renal excretion of porphyrin imparts an amber-brown discoloration to the urine (porphyrinuria) which emits a red fluorescence under ultraviolet light. Type II photodermatitis occurs due to deposition of porphyrins in the skin.
Congenital erythropoietic protoporphyria of cattle differs from bovine congenital porphyria in that affected animals develop only photodermatitis; discoloration of the teeth and bones, anemia, and porphyrinuria are not observed. This autosomal recessive disorder has been described in Limousine cattle, and results from a deficiency of ferrochelatase leading to accumulation of protoporphyrin IX in circulation and tissues.
Photodermatitis in animals and humans with erythropoietic porphyria results from accumulation of photodynamic pigments in the skin, which absorb ultraviolet and visible light and transform it into light of longer wavelength (red and infrared). The photoactive pigments cause production of reactive oxygen metabolites in the skin, either directly through transfer of energy to oxygen within the cytosol, or indirectly through activation of xanthine oxidase by calcium-dependent proteases. Oxygen free radicals then cause lipid peroxidation of cell membranes, rupture of lysosomes and mitochondria, complement activation, degranulation of mast cells, and release of vasoactive factors.
In addition to photodermatitis, variably severe hepatic disease may occur in human cases of protoporphyria, sometimes manifested as rapidly progressive liver failure associated with accelerating photosensitivity and cholestasis. Protoporphyrin IX, an hydrophobic porphyrin, is cleared from the serum by the liver and secreted in the bile where it enters the enterohepatic circulation. Cholestasis results from intracellular and canalicular precipitation of protoporphyrin, and is associated with inhibition of canalicular sodium/potassium ATPase. Hepatotoxicity, biliary hyperplasia, and portal fibrosis result.
Contributor: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500, and The Institut Pasteur, Unite de Genetique des Mammiferes, 25 Rue du Docteur Roux, 757244, Paris Cedex 15, France.
1. Montagutelli X: The ferrochelatase deficiency (Fechm1Pas) mutation, chromosome 18. In: Handbook of Mouse Mutations with Skin and Hair Abnormalities, Sundberg JP, ed., pp. 247-251, CRC Press Inc., Boca Raton, FL, 1994.
2. Jones TC, Hunt RD, King NW: Mineral deposits and pigments. In: Veterinary Pathology, 6th ed., pp. 73-75, Williams and Wilkins, Baltimore, MD, 1997.
3. Yager JA, Scott DW: The skin and appendages. In: Pathology of Domestic Animals, Jubb KVF, Kennedy PC, Palmer N, eds., 4th ed., vol. 1, pp. 595-596, Academic Press, San Diego, CA, 1993.
4. Cotran RS, Kumar V, Collins T: The skin. In: Robbins Pathologic Basis of Disease, 6th ed., pp. 1205-1206, WB Saunders, Philadelphia, PA, 1999.
5. Cox TM, Graeme JM, Alexander MD, Sarkany RPE: Protoporphyria. Seminars in Liver Disease 18:85-93, 1998.

Case IV - AP#2489 (AFIP 2641824)

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Signalment: Eleven-month-old, female, Watanabe hyperlipidemic (WHHL) rabbit.
History: One of four rabbits (three females and one male) used in a cyclosporine dose response study. The animal was given cyclosporine (Sandimmune I.V. ä) subcutaneously once daily at a dosage of 10 mg/kg. Blood cyclo-sporine levels were monitored once a week, along with blood urea nitrogen and creatinine levels. Approximately one month after beginning cyclosporine treatment, all animals began exhibiting signs of decreased food consumption, mild dehydration, and weight loss. The animal was euthanized two months following daily cyclosporine treatments.
Gross Pathology: All regions of the mammary gland were thickened and edematous.
Case 19-4. Gross Image. This closeup of mammary gland illustrates the nodularity and high fibrous connective tissue (white) content of these hyperplastic glands.
Laboratory Results: No abnormalities were detected in blood urea nitrogen and creatinine levels.
Contributor's Diagnosis and Comments: Hyperplasia, diffuse, marked, ductal and acinar, mammary gland.
Cyclosporine (cyclosporin A, CsA) is a potent immunosuppres-sive agent widely used in humans for preventing rejection of organ transplants and as treatment for autoimmune diseases. Notable side effects of chronic cyclosporine administration, such as nephrotoxicity and hepatotoxicity, have been well documented. Although rare, gynecomastia has been observed in male patients on cyclosporine therapy and is thought to occur as a result of an imbalance in the peripheral testosterone-to-estrogen ratio. This suggests that cyclosporine may cause endocrine dysfunction. Development of breast fibroadenomas has also been reported in women treated with cyclosporine.
Little work has been done to elucidate potential side effects of cyclosporine on endocrine ovarian function. Cyclosporine has been demonstrated to decrease plasma progesterone levels and augment the action of follicle stimulating hormone (FSH) in rats and rabbits. Moreover, studies have shown that the WHHL rabbit has an abnormal hypothalamic-pituitary-ovarian axis. Normally, stimulation and growth of the mammary gland is under hormonal control of both progesterone and estrogen. In the dose response study from which this case was derived, all females had marked mammary gland enlargement on post-mortem examination, but similar lesions were not observed in the male rabbit. Diffuse hyperplasia of acini and ductules and moderate desmoplasia were observed in histologic sections. The microscopic lesions strongly suggest that the inciting cause reflects ovarian hormonal dysfunction due to long term cyclosporine administration and/or strain characteristics unique to the WHHL rabbit.
Clinical signs observed during the course of this study (e.g., weight loss, decreased appetite) correlate to a toxic syndrome which has been reported in rabbits treated with cyclosporine.
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Case 19-4. Mammary gland. Glandular acini are hyperplastic, multifocally ectatic, and separated by increased amounts of loose fibrous connective tissue stroma.
AFIP Diagnosis: Mammary gland: Hyperplasia, ductal and stromal, diffuse, moderate to marked, WHHL rabbit, lagomorph.
Conference Note: In cats, mammary fibroepithelial hyperplasia, also known as fibroadenoma and fibroadenomatous hyperplasia, is a condition of young queens, usually less than two years of age, characterized by benign, nonneoplastic proliferation of ducts and periductal connective tissue in multiple glands. The condition also occurs in cats treated with certain medications. While the condition in cats shares several histologic features with the mammary gland of this rabbit, the etiology is endogenous progesterone or exogenous compounds that have progesterone-like activity; the condition regresses spontaneously following ovariohysterectomy or discontinuation of medical therapy.
In rabbits, cyclosporine seems to exert a negative effect on progesterone levels. In an experimental study, the ovaries of rabbits treated with cyclosporine prior to mating and during pregnancy contained fewer corpora lutea and had lower serum progesterone levels than control animals. In rat granulosa cells treated with cyclosporine in vitro at dosages designed to approximate immunosuppressive therapy, the drug augmented estrogen production at lower doses and was inhibitory at higher doses, while progesterone production was either unaffected or inhibited. These findings suggest that cyclosporine therapy may directly influence the steroidogenic function of the ovary, and thus affect the physiological state of target tissues of the sex steroids, such as the mammary gland; the exact mechanisms are not completely understood.
The WHHL rabbit has an aberrant hypothalamic-pituitary-ovarian axis, and it may be difficult to separate the role of this condition from that of cyclosporine in the pathogenesis of mammary hyperplasia. This rabbit serves as a model for familial hypercholesterolemia due to a defect in LDL receptor function which alters cholesterol availability to cells. Cholesterol is the precursor for steroid hormone synthesis, and a defective LDL receptor alters steroidogenesis. The low density lipoprotein particle is especially important for ovarian steroid synthesis in the corpus luteum, and reduced availability of cholesterol leads to significant reduction in plasma progesterone concentrations.
As noted by the contributor, cyclosporine is known for its potential nephrotoxicity and explains the monitoring of blood urea nitrogen and creatine levels in these rabbits during the course of the study. Chronic administration of cyclosporine at immunosuppressive doses to protect against organ transplant rejection may cause renal dysfunction due to decreases in glomerular filtration rates (GFR), decreased renal perfusion, increased secretion of renin, and activation of the renin-angiotensin system. Decreases in GFR result from afferent arteriolar degeneration. Lesions observed in the afferent arterioles consist of endothelial swelling, medial hyalinosis, and degeneration of the smooth muscle vascular wall. Very few compounds induce such specific lesions in specialized blood vessels. Other renal lesions caused by cyclosporine include vacuolation of the tubular epithelium, cortical interstitial fibrosis, and perivascular sclerosis of the hilar and interlobular arteries and arterioles, leading to glomerulosclerosis.
Contributor: Center for Comparative Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
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4. Al-Chalabi HA: Effect of cyclosporine A on the morphology and function of the ovary and fertility in the rabbit. International Journal of Fertility 29:218-223, 1984.
5. Robins ED, Nelson LM, Hoeg JM: Aberrant hypothalamic-pituitary-ovarian axis in the Watanabe heri-table hyperlipidemic rabbit. Journal of Lipid Research 35:52-59, 1994.
6. Gratwohl A, Riederer I, Graf E, Speck B: Cyclosporine toxicity in rabbits. Laboratory Animals 20:213-220, 1986.
7. Calne RY, et al.: Cyclosporine-A in clinical organ grafting. Transplantation Proceedings 13:349-358, 1981.
8. Robertson JL: Chemically induced glomerular injury: A review of basic mechanisms and specific xenobiotics. Toxicol Pathol 26:64-72, 1998.
9. Jones TC, Hunt RD, King NW: Genital system. In: Veterinary Pathology, 6th ed., pp. 1191-1200, Williams and Wilkins, Baltimore, 1997.
Conference Coordinator:
Ed Stevens, 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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