AFIP Wednesday Slide Conference - No. 8
21 October 1998

Conference Moderator:
Dr. D. Earl Green, Diplomate, ACVP
NCRR LSS SSB, Bldg. 28A, Room 117
28 Library Drive, MSC 5210
Bethesda, MD 20892-5210

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Case I - MK98-2222 (AFIP 2639069)
Signalment: Adult, male, cynomolgus macaque (Macaca fascicularis)
History: This monkey was part of an experiment investigating the effects of intraocular inoculation of an adenovirus. At termination of the scientific study, the animal was humanely euthanized via administration of intravenous barbiturates, the tissues of interest were collected, and the carcass was placed in refrigeration. A complete necropsy was performed 48 hours later. Incidental lesions were found in the lungs.
Gross Pathology: The macaque was well hydrated and well muscled with adequate stores of yellow fat. Along the ventral margins of the left cardiac and diaphragmatic lung lobes there were multiple 2-4 mm diameter spherical, yellowish-white, hard nodules in the pleura. Some nodules were attached to the pleura by a thin fibrous tag, while others caused adhesions between the parietal and pulmonary pleura. The mandibular and tracheobronchial lymph nodes were slightly enlarged and homogeneously gray. The heart, liver, kidneys, spleen, pancreas, gastrointestinal tract, and reproductive tract appeared normal.
Laboratory Results: Acid-fast stains of two mineralized pleural nodules and two tracheobronchial lymph nodes were negative.

Contributor's Diagnosis and Comments:

Morphologic diagnosis: Granulomata, verminous, subpleural, multifocal, mild, chronic.

Etiologic diagnosis: Pulmonary mesocestodiasis (pulmonary tetrathyridiosis).

Etiology: Larval Mesocestoides species (tetrathyridia).
The lung has multiple, discrete, subpleural granulomas which each contain a single immature cestode that lacks a bladder wall. The cestode larvae are nonsegmented and contain an invaginated scolex. Occasional sections of the larvae show the presence of suckers in the invaginated canal. Larvae have multiple small lightly basophilic calcareous corpuscles. The reaction is characterized by fibrous septal thickening, type II pneumocyte hyperplasia and a mix of inflammatory cells consisting of eosinophils, macrophages, plasma cells, and lymphocytes. A layer of degenerate inflammatory cells lies adjacent to the cestodes. The pleura is thickened by chronic pleuritis.
Differential diagnosis includes Diphyllobothrium species, Mesocestoides species, and Echinococcus species. Because the cestodes lack bladder walls, Echinococcus (hydatid cysts) was ruled out. Histologically, the invaginated scolex, absence of a bladder wall, and the 4 suckers are consistent with Mesocestoides.
Mesocestoides species are members of the order Cyclophyllidea and are found in many parts of the world, including Africa, Asia and the United States. They can live in a wide range of hosts, but are particularly widespread in carnivores. The life cycle of Mesocestoides is incompletely understood and still largely unknown, but there are thought to be two metacestode stages and two intermediate hosts. The first stage metacestode probably occurs in a coprophagous arthropod, with the second stage metacestode occurring in a vertebrate. Mammals and reptiles are not infected directly by eggs, but must ingest the larval form of the cestode in the first intermediate host.
The larval form of Mesocestoides is known as a tetrathyridium. Tetrathyridia are flat, nonsegmented and contractile, 2 to 70 millimeters long, and resemble fine connective tissue strands. In an unsuitable host, the tetrathyridia persist in an encapsulated form until they are ingested by a definitive host. Once ingested by a definitive host, the tetrathyridia develop to adults in approximately 21 to 30 days. The adult worm is 30 to 150 centimeters long and a maximum of 3 millimeters wide. It has an unarmed scolex with 4 suckers and no rostellum. Each proglottid has one set of male and female reproductive organs with a single genital pore characteristically placed ventromedially. One feature unique to Mesocestoides is the method used for asexual reproduction. Other cestodes reproduce asexually by budding, but Mesocestoides are able to reproduce asexually by developing a longitudinal fissure that begins at the scolex.
Generally, the larval cestodes are either encapsulated in the retroperitoneal and subcutaneous tissues or are free in the abdominal and thoracic cavities. There are reports of tetrathyridia occurring in the scrotum, bladder, liver, intestines, and rarely in the lungs of mammals, reptiles, birds and amphibians. When the infection occurs in the pleural spaces, the cestodes are generally free floating. Mesocestoides are seldom found in nonhuman primates, and are exceptionally rare in macaques. When encountered in macaques, the tetrathyridia are either free in the abdomen or encysted in the mesentery, and rarely involve the lung. Previous reports have referred to the chronic infection as "pearl disease" in primates because of the size, shape and color of the verminous granulomas.
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Case 8-1 . Lung. The 2x view shows 3 profiles of cestode larva surrounded by a dense inflammatory infiltrate forming a granuloma adjacent to the pleura surface. Surrounding alveoli have mild alveolar emphysema and thickened alveolar septa. At 40x, multiple calcarious corpuscles (blue bodies with clear halos) are scattered within the mesenchyme of the larva. The tegument has a thick eosinophilic, 15-20u, smooth surfaced cuticle. The surrounding interstitium is fibrotic with moderate numbers of infiltrating eosinophils and fewer macrophages and lymphocytes.
AFIP Diagnosis: Lung: Granulomas, eosinophilic, multiple, with larval cestodes, cynomolgus monkey (Macaca fascicularis), nonhuman primate, etiology consistent with Mesocestoides sp.
Conference Note: Conference participants identified a cestode parasite characterized by a thick tegument, the presence of calcareous corpuscles within a parenchymatous body, and an invaginated unarmed scolex; a few sections contained identifiable suckers. Muscle cells divide the cortex from the medulla, identifying the cestode as a cyclophyllidian, rather than a pseudophyllidian such as a sparganum. Additionally, spargana contain a bothrial slit without suckers. Also included in the differential diagnosis discussed by conference participants was the coenurus form of taeniid metacestodes. Coenuri in tissue sections occur as single or loculated fluid-filled cysts in which many nodular invaginated scolices are present in clusters on the inner wall. The inverted scolices also contain suckers, and in all species, except Coenurus bovis, hooks may be found on the rostellum.
In most instances, the scolex of Mesocestoides sp. is invaginated when examined in tissue sections. During collection of live tetrathyridia for use in feeding experiments, the cestode larvae are freed from tissue nodules by placement into digestive juices. After release into the medium, the scolex of the tetrathyridium may be induced to evaginate by subjecting the larva to alternating temperatures between an incubator (37 °C) and refrigerator (4 °C).
Traditionally, determination of species within the genus Mesocestoides has relied on inoculation of encysted and free tetrathyridia into definitive hosts and sacrificing the animals after tapeworm proglottids become evident in the feces of the inoculated subjects. Identification of the cestode species is based upon the morphology of adult tapeworms harvested from the definitive hosts at necropsy. In practice, however, these feeding experiments are not always successful or feasible.
Recently, tapeworm larvae were identified in dogs with peritoneal infections caused by Mesocestoides sp. using polymerase chain reaction amplification of cestode DNA. While the protocol did not determine the exact species of Mesocestoides infecting the dog, refinement of the technique may eventually eliminate the necessity of feeding trials for parasite speciation.
There is abundant proteinaceous fluid filling alveoli in some sections of lung. This fluid is considered artifact caused by refrigeration of the carcass and the effects of intravenous barbiturate euthanasia. Tissue sections may have originated from dependent areas of lung. There were no clinical signs of pulmonary edema, and the microscopic features suggestive of antemortem edema, such as dilated lymphatics, are not evident in the examined sections.

Contributor: Pathology Unit, Veterinary Resources Program, NCRR, National Institute of Health, 9000 Rockville Pike, Bethesda, MD 20892.

1. Sasseville VG, et al.: A case of pulmonary cestodiasis in a simian immunodeficiency virus-infected pigtailed macaque (Macaca nemestrina) in which virus-infected leukocytes are present within the lesion. J Med Primatol 25:251-256, 1996.
2. Fincham JE, et al.: Pleural Mesocestoides and cardiac shock in an obese vervet monkey (Cercopithecus aethiops). Vet Pathol 32:330-333, 1995.
3. Hubbard GB, et al.: Mesocestoides infection in captive olive baboons (Papio cynocephalus anubis). Lab Anim Sci 43:625-627, 1993.
4. Guillot LM, Green LC: Pulmonary cestodiasis in a cynomolgus monkey (Macaca fasicularis). Lab Anim Sci 24:158-160, 1992.
5. Schmidt GD, Roberts LS: Tapeworms. In: Foundations of Parasitology, pp. 373-398, Times Mirror/Mosby College Publishing, St. Louis, Missouri, 1985.
6. Reid WA, Reardon MJ: Mesocestoides in the baboon and its development in laboratory animals. J Med Primatol 5:345-352, 1976.
7. Wong MM, Conrad HD: Parasitic nodules in the macaques. J Med Primatol 1:156-171, 1972.
8. Houser WD, Paik SK: Hydatid disease in a macaque. J Amer Vet Med Assoc 159:1574-1577, 1971.
9. Specht D, Voge M: Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. J Parasitol 51:268-272, 1965.
10. Barker IK, Van Dreumel AA, Palmer N: Infectious and parasitic diseases of the gastrointestinal tract. In: Pathology of Domestic Animals, Jubb KVF, Kennedy PC, Palmer N, eds., 4th ed., vol. 2, pp. 287-292, Academic Press, 1993.
11. Crosbie PC, et al.: Diagnostic procedures and treatment of eleven dogs with peritoneal infections caused by Mesocestoides spp. J Amer Vet Med Assoc 213:1578-1583, 1998.
Case II - 97F377 (AFIP 2642181)
Signalment: Nine-month-old, Nile tilapia, male, piscine, Tilapia nilotica.
History: This tilapia was one of several submitted for diagnostic evaluation following an outbreak of disease in a large indoor recirculating aquaculture facility. Mortalities approaching 20% were occurring throughout the facility, in both fingerling and grow-out tanks. Despite the mortalities, a large portion of the fish population continued to feed aggressively. The primary clinical signs reported by the producer were exophthalmia, cloudy eyes and aimless circling near the water's surface. With the exception of high dissolved carbon dioxide levels, water quality parameters were within acceptable limits. The producer reported periodic spikes in nitrite levels and low dissolved oxygen levels in some tanks during the period leading up to the initial mortalities.

Gross Pathology: Although gross lesions varied between individuals, this particular tilapia, weighing 0.75 lb (0.34 kg), exhibited changes representative of the diseased population as a whole. The fish was submitted live, euthanized, and necropsied immediately. There was generalized darkening of the skin accompanied by reddening of the pectoral fin bases. Severe exophthalmia, corneal opacification, and cloudy intraocular exudate were present bilaterally. The abdomen was distended by copious amounts of slightly opaque watery fluid containing flecks of fibrin. The liver, spleen and kidney were diffusely pale. Splenomegaly was marked, and fibrinous material was loosely adhered to its capsular surface. The pericardial chamber also contained a cloudy exudate, and fibrin was adhered to the epicardium. The meninges were congested, and cerebrospinal fluid appeared excessive and slightly opaque. Additional lesions present in other fish included subcutaneous abscesses, particularly in the caudal peduncle, at the base of the pectoral fins, and along the ventral margin of the mandible.
Case 8-2. Gross Viscera. Shows cloudy opacity of anterior chamber of eye (ophthalmitis), splenomegaly, and fibrin tags on the splenic capsule (fibrinous peritonitis) and heart (fibrinous epicarditis). Gills have been removed (note stumps of branchial arches). The branchial chamber is partly coated with a tan (fibrinous) exudate. The kidney (?) is very pale and/or covered with fibrin.

Laboratory Results: Streptococcus iniae was isolated in pure culture from the brain, liver and spleen.
Contributor's Diagnoses and Comments:
1. Brain, meningitis, granulomatous, moderate, with intralesional cocci.
2. Membranous labyrinth, otitis interna, granulomatous, moderate to severe.
Etiology: Streptococcus iniae.
Streptococcus iniae was first reported in the United States in 1994 from a Texas tilapia farm experiencing 75% mortalities. The disease then quickly surfaced around the United States, probably due to rapid expansion of the industry and the movement of infected fingerlings around the country. Since that time, it has become the most significant disease problem facing the tilapia industry. The disease has subsequently been reported in hybrid striped bass cultured in association with infected tilapia. Cases of bacterial meningoencephalitis in tilapia and rainbow trout appeared in Israel in 1986. The offending agent was given the new species name of S. shiloi and was not properly identified as S. iniae until 1995. Streptococci, with biochemical properties similar to S. iniae, have been isolated from cultured tilapia and other fish species in Japan dating back to 1979.

Streptococcus iniae is a b-hemolytic coccus first isolated from a captive Amazon River dolphin in 1976. The bacterium is not classifiable by the traditional Lancefield typing system, and dolphin biotypes are reportedly non-pathogenic to fish. In the experience of this lab, cases of S. iniae present as either an acute fulminating septicemia or in a more chronic form limited to the central nervous system. In the acute septicemic form, gross lesions, including any or all or those described above, may be present. Microscopic lesions include meningoencephalitis, perineuritis, polyserositis, epicarditis, myocarditis, and cellulitis. Cocci can sometimes be seen in hematoxylin & eosin stained sections, but are readily evident when stained with tissue Gram stains. In these cases, the bacteria can be readily isolated from most organs and serous membranes in large numbers on blood or brain heart infusion agars. In chronic cases, small effete granulomas with caseous centers may be present in parenchymatous organs, but active inflammation is usually limited to the central nervous system. Typically, the organism can be cultured only from the brain in these cases.
Infections are primarily a problem in closed recirculating culture systems and are much less common in ponds. This is probably related to the higher stocking densities and problems of water quality (such as high nitrite levels) encountered in recirculating culture systems. This would suggest that stress may play a role in outbreaks of disease. While typically susceptible to oxytetracycline, potentiated sulfonamides and ampicillin, treatment failures using medicated feeds are common. Depopulation of affected facilities, disinfection and restocking with disease free fish are currently the best means of eliminating the organism. During the winter of 1995-1996, invasive infections due to S. iniae were reported from the Toronto area in human patients who had suffered skin injuries while handling fresh fish, including tilapia. Infections included cellulitis of the hand and one case of endocarditis. All cases were successfully treated with antibiotics.
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Case 8-2. Otic vesicle, Brain. The otic vesicle is heavily infiltrated by inflammatory cells and fibrin, and contains a mineralized otolith. The connective tissue between the brain and otic vesicle is similarly infitrated by abundant inflammatory cells.
Brown & Brehn, 40x obj
Case 8-2 . Skull. Some macrophages in the exudate surrounding the otic vesicle contain abundant Gram positive cocci.
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Case 8-2.Gill filaments and lamella. The interlamellar space is partially filled between most gill lamella by an infiltrate of macrophages, lymphocytes, and fewer mucus cells and/or epithelial cells.
AFIP Diagnoses:
1. Brain; meninges; otic labyrinth; pericardium; and skeletal muscle: Inflammation, histiocytic and lymphoplasmacytic, multifocal, moderate, with intrahistiocytic cocci, Nile tilapia (Tilapia nilotica), piscine.
2. Gill: Branchitis, lymphoplasmacytic, multifocal, mild, with epithelial hyperplasia.
Conference Note: Epicarditis, choroiditis, and ventriculitis are present in some sections. Additionally, some sections contain amphophilic, amorphous bodies within the otolabyrinth; these may be otoliths.
Conference participants found the intense histiocytic inflammatory response to this streptococcal organism rather unique. Streptococcal infection is typically associated with an intensely neutrophilic, suppurative inflammatory response. In one report of experimental and field infections (S. difficile and S. shiloi) in cultured fish, the primary microscopic changes were in the brain and consisted of subarachnoid hemorrhages and parenchymal mononuclear inflammatory infiltrates with giant cells.
A report of granulomatous valvular endocarditis is described in humans in which Streptococcus viridans, an alpha-hemolytic streptococus, was cultured and identified histologically in diseased aortic valves in two individuals. Fibrinoid necrosis and histiocytic granulomas were observed histologically, and streptococci were found in the cytoplasm of macrophages. Another unique, though dissimilar, lesion has been reported in streptococcal bacteremia in Singapore house geckos. Histomorphologic changes were described as multiple, nodular, expansile colonies of cocci primarily affecting the lungs and kidneys, often in the absence of an inflammatory reaction. The minimal inflammatory infiltrates that were present consisted of occasional macrophages, lymphocytes, and rare granulocytes at the edges of the bacterial colonies. Chains of cocci were embedded within an amphophilic, homogenous material, identified ultrastructurally as a thick peptidoglycan layer and a thinner glycocalyx surrounding the bacterial cell wall. In rats, local or systemic injection of peptidoglycan-polysaccharide polymers from the bacterial cell walls of group A streptococci leads to acute inflammation which can develop into chronic, spontaneously relapsing granulomatous inflammation in several organs. The histiocytic inflammatory response in tilapia infected with S. iniae may be a function of the immune response in fish and other poikilothermic animals, coupled with the structural and biochemical properties of non-Lancefield streptococci which may incite an atypical inflammatory response.
Whether recent human infections of S. iniae represent a new zoonotic pathogen or cases of previously unrecognized disease remains unknown. Cellulitis in humans occurring spontaneously or associated with injury is most often due to S. pyogenes or Staphylococcus aureus, and cultures of such lesions are usually nondiagnostic or not performed. Under certain circumstances, the beta-hemolysis of S. iniae on culture media may not be evident, and it may be considered a contaminant. The reports of human infection seem to coincide with the recent identification of the organism in cultured fish and support the possibility that S. iniae may be a newly emerging pathogen.
Another streptococcal species capable of producing zoonotic infection is Streptococcus suis. Outbreaks of S. suis septicemia and meningitis occur in pigs, especially under adverse environmental conditions. Human infections with S. suis have been described in individuals handling live or slaughtered infected pigs. The portal of entry for the organism is thought to be the skin. Human infections with S. suis and S. iniae seem to share several clinical and epidemiological similarities. Changes in production, storage, distribution, and preparation of food may provide increased opportunity for human exposure to pathogenic organisms.
Contributor: Department of Pathology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803.
1. Perera RP, et al.: Streptococcus iniae associated with mortality of Tilapia nilotica x T. aurea hybrids. J Aquatic Animal Health 6:335-340, 1994.
2. Eldar A, Bejerano Y, Livoff A, Horovitcz A, Bercovir H: Experimental streptococcal meningoencephalitis in cultured fish. Vet Microbiol 43:33-40, 1995.
3. Eldar A, Berejano Y, Bercovier H: Streptococcus shiloi and Streptococcus difficile: Two new streptococcal species causing a meningoencephalitis in fish. Cur Microbiol 28:139-143, 1994.
4. Weinstein MR, et al.: Invasive infections due to a fish pathogen, Streptococcus iniae. New Eng J Med 337:589-594, 1997.
5. Gassel AM, et al.: [Granulomatous endocarditis caused by streptococcus]. Pathologe 15:40-43, 1994.
6. Sartor RB, Herfarth H, Van Tol EAF: Bacterial cell wall polymer-induced granulomatous inflammation. Methods 9:223-247, 1996.
7. McNamara TS, Gardiner CH, Harris RK, Hadfield TL, Behler JL: Streptococcal bacteremia in two Singapore house geckos. J Zoo Wildl Med 25:161-166, 1994.
Case III - 7261-P-1 (AFIP 2637890)
Signalment: Four-year-old, female baboon (Papio cynocephalus/anubis).

History: The animal received an autologous stem cell transplant 24 hours following irradiation with 1020cGy of radiation over two days (split 510cGy each day). At approximately 25 days post-engraftment (28 days post-irradiation), the animal presented with primary complaint of hematuria, anemia, leukopenia and thrombocytopenia.
Contributor's Diagnoses and Comments:
1. Kidney:
a. Tubular degeneration and necrosis, hemoglobinuric, multifocal, mild to moderate.
b. Glomerulopathy, membranous, global, mild to moderate.
c. Nephritis, lymphoplasmacytic, fibrosing, multifocal, mild to moderate with multifocal hemorrhage.
2. Large intestine:
a. Hemorrhage, proprial and submucosal, coalescing, severe, acute.
b. Arteriopathy and periarteriolar fibrosis, submucosal, multifocal, mild.

3. Heart:
a. Cardiac fibrosis, moderate with mild multifocal lymphocytic epicarditis.
b. Epicardial and endocardial hemorrhage, multifocal, mild, acute.
Etiology: Erythrocytic parasitism (suggestive of Babesia sp.).
This animal's acute decline and nephropathy were interpreted to be associated with a hemolytic crisis secondary to erythrocytic parasitism. The morphology of the erythrocytic parasite was most suggestive of babesiosis. This infection has been previously documented in baboons (1) and has previously been recognized in animals undergoing a similar protocol of irradiation, bone marrow transplantation or repeated blood transfusion in our facility. The organism can be difficult to appreciate in the submitted sections. The submitted 2x2 color transparency illustrates the organisms at 100X.
This infection was facilitated by immunosuppression secondary to total body irradiation and failure of the subsequent stem cell engraftment. Numerous chronic lesions in the submitted sections of kidney, heart and large intestine can be attributed to this irradiation (2,3). These include glomerulopathy, mild fibrosing nephritis, mild arteriopathy (characterized by mild hyaline changes in arteriolar media and periarterial fibrosis), multifocal cardiac fibrosis, and multifocal hemorrhage (secondary to thrombocytopenia).
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Case 8-3. Kidney. One or more refractile protozoa are found within many RBCs of congested interstitial capillaries. There is fibrillar to amorphous proteinaceous material within a collecting duct.
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Case 8-3 . Kidney. The glomerulus contains increased numbers of mesangial cells which compress glomerular capillaries. Adjacent tubules are filled with eosinophilic material forming hyaline casts.
Movat Stain, 4x obj
Case 8-3. Cardiac muscle. Myocardial muscle is partly replaced by fibrous connective tissue (fibrosis).
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Case 8-3. Colon. The lamina propria is markedly expanded by diffuse hemorrhage which distorts and separates the mucosa from the underlying tunica muscularis.
AFIP Diagnoses:
1. Kidney: Tubular epithelial degeneration and necrosis, multifocal, mild to moderate, with intratubular proteinaceous casts and erythrocytic debris, and intraerythrocytic piroplasms, baboon (Papio cynocephalus/anubis), nonhuman primate.
2. Kidney; heart, atrium; large intestine (per contributor), arteries: Fibrinoid necrosis, multifocal, with perivascular hemorrhage and fibrin deposition.
3. Kidney: Glomerulopathy, membranous, global, multifocal, mild to moderate.
4. Heart, atrium: Contraction band necrosis, multifocal.
5. Heart, atrium: Fibrosis, multifocal, mild.
6. Large intestine, lamina propria (per contributor): Hemorrhage, diffuse, severe.
Conference Note: This case was studied in consultation with the Departments of Cardiovascular Pathology and Genitourinary Pathology. Small arteries within the atrium have swollen endothelium, and there is expansion of the tunica media by hyalinized eosinophilic material, interpreted as fibrinoid change. Fibrin deposition is present within the adventitia of some vessels. Myocyte contraction band necrosis is also present, as are multifocal areas of fibrosis. Similar arterial changes are present within the kidney. Many glomeruli are hypercellular and contain increased amounts of mesangial matrix; global and segmental synechiae are present in some glomeruli. Severe hemorrhage is observed in the lamina propria of the large intestine, making tissue identification difficult.
Multifocally, some kidney tubules are filled by an eosinophilic homogenous material interpreted as proteinaceous fluid, while a few tubules contain fibrin and sloughed epithelial cells. Still other tubules contain moderate amounts of a more brightly eosinophilic, globular to crystalline material interpreted to be hemoglobin. Protein droplets occur within tubular epithelial cells, especially within proximal convoluted tubules. Histochemical stains performed at the AFIP demonstrate abundant iron within tubules and in tubular epithelial cells.
The histologic lesions in this baboon can be briefly summarized as membranous glomerulopathy, vascular fibrinoid change in the heart and kidney, severe hemorrhage in the lamina propria of the large intestine, and hemoglobinuric nephrosis. Conference participants found it difficult to separate the microscopic lesions of irradiation from those of babesiosis. Membranous glomerulopathy, hemorrhage in the large intestine, and vascular changes observed in several organs are most consistent with irradiation effect; however, a contribution to the vascular lesions by concurrent babesiosis cannot be excluded. Hemoglobinuric nephrosis, as evidenced by extensive hemoglobin pigment within tubules and tubular epithelium, is not related to radiation, and is most likely secondary to parasite-induced hemolysis.
The vascular changes are characteristic of radiation effect. In general, the entire vessel is affected by irradiation, although the endothelium is the most sensitive part. The pathologic effects on endothelial cells become evident when they attempt to divide, usually 3-4 weeks post-irradiation. The old endothelium fails to become replaced by new endothelial cells, leading to exposure of the underlying basement membrane, consumption of platelets, thrombocytopenia, and hemorrhage. Decreased or absent platelet production due to irradiation-induced bone marrow suppression exacerbates the thrombocytopenia and hemorrhage.
Babesiosis is a tick borne disease caused by microorganisms of the genus Babesia. Transfusion transmitted infections may also occur. There are approximately 100 known species which infect a wide variety of animals, including cattle, horses, dogs, cats, swine, sheep, donkeys, goats, raccoons, skunks, fowl, wild rodents and ruminants, and monkeys. The pathogenicity of babesia is directly related to the species and strains infecting a given animal. Host age and immunologic response generated against the parasite are also factors in the disease. Hemolytic anemia and multiple organ dysfunction account for most of the clinical signs observed in animals with babesiosis. Disseminated intravascular coagulation can be a fatal complication in canine babesiosis. Tissue hypoxia due to anemia, shock, and vascular stasis, results in release of cytokines, and widespread inflammation and damage to multiple organs. In experimentally infected dogs, hypoxia appears to be more important than hemoglobinuria in producing damage to the kidneys.

Contributor: Department of Comparative Medicine, University of Washington, Seattle, WA 98195-7190.
1. Weyhrich JT, et al.: Piroplasmosis in transfused, immunosuppressed baboons. Lab Anim Sci 43:390, 1993.
2. Mostofi FK, Berdjis CC: Radiopathology of the kidney. In: Pathology of Irradiation, Berdjis CC ed., pp. 597, Williams & Wilkins Co., Baltimore, MD, 1971.
3. Berdjis CC: The cardiovascular system. In: Pathology of Irradiation, Berdjis CC ed., pp. 377, Williams & Wilkins Co., Baltimore, MD, 1971.
4. Taboada J: Babesiosis. In: Infectious Diseases of the Dog and Cat, Green CE ed., 2nd ed., pp. 473-481, W.B. Saunders, Philadelphia, PA, 1998.
Case IV - U2579 (AFIP 2641488)
Signalment: Two-year-old Atlantic salmon (Salmo salar).
History: This fish was from a population reared in sea water net pens held in the Bay of Fundy, New Brunswick, Canada. The population at risk was experiencing steadily increasing mortality over several weeks. Clinical signs included anorexia, inability to hold position in the water column, and respiratory distress (listlessness).

Gross Pathology: Unilateral to bilateral exophthalmos, marked petechial to suffusive ventral hemorrhage, and mild branchial pallor were prominent external signs. Internally, affected fish had moderate amounts of serosanguinous to hemorrhagic peritoneal fluid, and congested lower intestines, pyloric caeca and spleens. Liver congestion was rarely observed (1/10), as were intramuscular, perivisceral and peritoneal petechiae. All fish were off feed. On section, copious amounts of blood flowed freely from the kidney.
Laboratory Results: Samples submitted from the population for bacteriology were negative. Clinical pathology/hematology was not completed on this group of fish, but previous evaluations of similarly affected populations showed evidence of marked anemia in some fish (Byrne et al., 1998). Virus isolation on the salmon head kidney (SHK) cell line produced cytopathic effect consistent with that previously reported for the orthomyxovirus known to be the causative agent of infectious salmon anemia (ISA) in Norwegian pen-reared Atlantic salmon (Dannevig et al., 1995). Confirmation of this finding was completed by IFAT using a monoclonal antibody to the ISA virus produced in Norway (Falk and Dannevig, 1995) and RT-PCR using primer series similarly identified by Norwegian workers (Mjaaland et al., 1997). Electron microscopy of gill tissue from field cases in New Brunswick (see 2x2 photo) revealed the presence of viral particles in lamellar capillary endothelium of appropriate size (90-120 nm) and morphology consistent with the orthomyxovirus identified as the causative agent of ISA in Norway (Hovland et. al., 1994; Nylund et. al., 1995).
Case 8-4. Electron micrograph of gill endothelium. Multiple oval shaped electron dense virus-like particles free in the cytosol appear to be budding from several profiles (right) of smooth endoplasmic reticulum (SER). Multiple mitochondria in the upper half of the image have mild expansion of the inner compartment.
Contributor's Diagnoses and Comments:
1. Hepatic necrosis, congestion and hemorrhage, multifocal, acute, moderate.
2. Renal interstitial congestion and hemorrhage, diffuse, acute, severe.
3. Renal tubular necrosis, multifocal, acute, moderate.
Etiology: Orthomyxovirus.

Primary histological lesions were noted in the liver and kidney. Liver pathology was characterized by a range of changes, from mild diffuse sinusoidal congestion to marked peliosis and hemorrhage, often accompanied by multifocal to bridging regions of coagulative necrosis. Affected regions of liver rarely contained a granulocytic infiltrate. In some fish, mild perivascular cuffing by a mixed leukocyte population was evident, but this was not a consistent finding. The kidney also presented with sinusoidal congestion and marked interstitial hemorrhage. In the more severely affected fish, multifocal acute tubular necrosis, with eosinophilic casting was also present and considered pathognomonic for the disease (Mullins and Groman, submitted; Byrne et. al., 1998). Other tissues (not present on slides) with congestion included filamental arterioles of the gill, splenic sinusoids, vasculature of the pyloric caeca/intestinal lamina propria, and visceral mesenteries. Erythrophagia was a consistent finding in all cases, especially prominent in the spleen.
Mortalities ascribed to infectious salmon anemia in the Bay of Fundy were first thought to have surfaced in the summer of 1996. At that time, however, pathology was primarily restricted to the kidney (as noted above), and the term hemorrhagic kidney syndrome (HKS) was adopted (Byrne et al., 1998). Significant effort was made, without success, during the autumn and winter of 1996-1997 to identify a cause for this syndrome, including submission of frozen sections to Norway for IFAT stain for the ISA virus. In the summer of 1997, with the aid of the Norwegian SHK cell line, ISA virus was finally isolated and confirmed as the virus causing mortality in fish from the Bay of Fundy.
It is noteworthy that, during this period, the pathological profile changed significantly to that presented here, especially in naive fish which entered sea water in the spring of 1997 and subsequently contracted the disease. The difference in pathological presentation between the ISA as seen in Norway (and more recently in Scotland) and that described in New Brunswick is significant enough to suggest that there are at least 2 strains of the virus, i.e. the Norwegian strain causing primarily liver pathology (Evensen et al., 1991; Speilberg et al., 1995), and the New Brunswick strain targeting the kidney.
ISA is currently a serious problem for the salmon farming industry in New Brunswick (Canada), Norway and Scotland, and efforts are underway to intensively survey salmon populations for the disease as well as develop a vaccine.
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Case 8-4. Kidney. Hematopoietic cells are largely replaced by hemorrhage and congestion which expands the interstitium and separates tubules.
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Case 8-4. Kidney. Multifocally, tubular epithelium is necrotic.
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Case 8-4. Multifocally and randomly throughout the hepatic parenchyma, there are pale zones of hepatocellular necrosis (loss of cell detail, karyolysis, pyknosis, & cell loss).
AFIP Diagnoses:
1. Trunk kidney: Congestion and hemorrhage, diffuse, moderate, Atlantic salmon (Salmo salar), piscine.
2. Liver, hepatocytes: Degeneration and necrosis, multifocal.
3. Kidney: Necrosis, tubular epithelium, multifocal.
4. Spleen: No significant lesions.
Conference Note: Infectious salmon anemia (ISA) is a viral disease of farmed salt water Atlantic salmon first reported in Norway in 1984. The etiologic agent of ISA is an enveloped RNA virus with structural characteristics consistent with an orthomyxovirus. The virus replicates in endothelial cells, endocardium, and leukocytes. The disease is characterized by severe anemia, leukopenia and high mortalities. Pathological changes include congestion of the liver, spleen and foregut, hemorrhagic liver necrosis, ascites, pale gills, and petechiae in the viscera.
In July 1996 increased moribund and dead farmed Atlantic salmon were observed on Canada's eastern seaboard with no identified etiology. Clinically, affected fish were lethargic, anorectic, and lacked external lesions. Clinicopathological findings included anemia, hypoproteinemia, hypernatremia, and hyperchloremia. Gross lesions found during necropsy included multifocal reddening of the kidney, pale gills, exophthalmos, ascites, and splenomegaly. The major histopathological findings were renal interstitial hemorrhage and acute tubular necrosis with tubular casts. The disease was termed hemorrhagic kidney syndrome (HKS) of Atlantic salmon. While a virus was suspected as the underlying cause, the etiology was not determined until late in 1997 when an orthomyxovirus was isolated from diseased Canadian Atlantic salmon at the New Brunswick laboratory.
The differences in the gross and histologic findings of ISA virus in Norwegian and Canadian salmon with HKS are probably multifactorial. The variation in Atlantic salmon stock, viral strain, water temperatures, infectious dose, age of fish, immune status, and concomitant infections may act as variables that influence the pathogenesis and clinicopathologic presentation of the disease. In addition to the kidney lesions of HKS described in 1996, Canadian Atlantic salmon have subsequently developed hepatic, branchial, and enteric lesions typical of ISA. Recent archival reviews of Norwegian case submissions of ISA identified the presence of renal lesions similar to those described in New Brunswick, Canada. Eventual comparison of the genomic sequences may help clarify potential differences in viral strains.
Contributor: Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI C1A 4P3, CANADA.
1. Byrne PJ, MacPhee DD, Ostland VE, Johnson G, Ferguson HW: Haemorrhagic kidney syndrome of Atlantic salmon, Salmo salar L. J Fish Dis 21:81-91, 1998.
2. Dannevig BH, Falk K, Namork E: Isolation of the causal virus of infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon head kidney. J Gen Virol 76:1353-1359, 1995.
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5. Hovland T, Nylund A, Watanabe K, Endresen C: Observation of infectious salmon anaemia virus in Atlantic salmon, Salmo salar L. J Fish Dis 17:291-296, 1994.
6. Mjaaland S, Rimstad E, Falk K, Dannevig BH: Genomic characterization of the virus causing infectious salmon anaemia in Atlantic salmon (Salmo salar L.): An orthomyxo-like virus in a teleost. J Virol 71:7681-7686, 1997.
7. Mullins J, Groman D: Infectious salmon anaemia in salt water Atlantic salmon in New Brunswick. Bulletin European Assoc Fish Pathol 18:110-116, 1998.
8. Nylund A, Hovland T, Watanabe K, Endresen C: Presence of infectious salmon anaemia virus (ISAV) in tissues of Atlantic salmon, Salmo salar L, collected during three separate outbreaks of the disease. J Fish Dis 18:108-111, 1995.
9. Speilberg L, Evensen O, Dannevig BH: A sequential study of the light and electron microscopic liver lesions of infectious anemia in Atlantic salmon. Vet Pathol 32:466-478, 1995.
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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