AFIP Wednesday Slide Conference - No. 17
January 19, 2000

Conference Moderator:
Dr. Jerry L. Quance, Diplomate, ACVP
Maryland Department of Agriculture
Animal Health Laboratory
Frederick, MD 21702
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Case I - UFSM-1 (AFIP 2687023)
Signalment: Six-month-old, Holstein Friesian, female, bovine.
History: This is one of four 6-month-old heifers housed in the same stall. There were 83 dairy cattle (Holstein Friesian) in this farm. This heifer had normal temperature, loss of appetite, apathy, incoordination, opisthothonus, and lateral recumbency. It was euthanatized 5 days after the onset of clinical signs. All cattle of this farm were tested with the skin mammal tuberculin test and 50% were positive. Four months after the death of this heifer, another 4-year-old cow died after showing similar neurological signs for 2 days. Gross lesions found in the cow were similar to those described here.
Case 17-1. Gross Brain (see comments in text).
Gross Pathology: The necropsy was performed at the farm by the submitting veterinarian. Reportedly, no gross changes were found in organs other than the brain. Gross examination of the brain revealed increased cerebrospinal fluid (CSF), coning of the cerebellum and flattening of cerebral gyri. The leptomeninges were markedly thickened mainly over the cerebellum, base of the cerebrum, and occipital cortex. The mesencephalic aqueduct was blocked by cellular and fibrin exudate and dilation of lateral ventricles (internal hydrocephalus). The fixed brain was sent to our laboratory.

Contributor's Diagnoses and Comments: Thalamus, hippocampus and rostral colliculi, granulomatous meningoencephalitis and encephalitis with Langhans giant cells, caseous necrosis, mineralization, vasculitis, and thrombosis.
Etiological diagnosis: Bacterial meningitis and encephalitis.

Etiology: Mycobacterium sp.
Morphological lesions are typical of bovine tuberculosis. Lesions are more pronounced in the meninges but extension into the brain through the vascular spaces occur. In some slides well developed granulomas are seen within brain substance. Granulomatous vasculitis associated with fibrinoid necrosis and occasional thrombi are observed. Ziehl Neelsen stain revealed acid fast organisms associated with these lesions. Although bacteriological culture was not performed, the distribution of lesions suggests that M. bovis is the most likely species of Mycobacterium involved.
Tuberculosis is endemic in Brazil. A survey carried out in 237 dairy herds showed that 0.66% of the tuberculin tested animals were positive. In another survey carried out in abattoirs in the state of Rio Grande do Sul, southern Brazil, in a period of 8 years, 0.64% of slaughtered beef cattle had gross lesions of tuberculosis. The prevalence of the disease is greater in the north of the country. In areas with a high prevalence, bovine tuberculosis is almost always caused by Mycobacterium bovis and only when the control of the disease is improved, M.avium-intracellulare is significant.
The pathogenesis of tuberculosis has been extensively studied. From a primary focus (known as primary complex) in calves the disease disseminates in different ways: (i) by extension, forming new tubercles; (ii) by lymphatics draining the primary complex to the regional lymph nodes; (iii) by retrograde lymphatic route, involving serosae; (iv) by lymphatic and hematogenous route, through access to the thoracic duct and vena cava, causing miliary tuberculosis, which is more common in the lung and less so in the liver, kidney, and brain; (v) by direct hematogenous route, which is common in the congenital form of tuberculosis, and (vi) by dissemination through the bronchi.
The distribution of the lesions of tuberculosis in the central nervous system predominates in the meninges of the base of the brain (basilar tuberculous meningitis). From there, the lesions may extend to the submeningeal nervous tissue. Most likely, the organism enters the brain by hematogenous route and is retained in the choroid plexus and meningeal vessels reaching the CSF and the ventricular system. Meningeal lesions are similar to those seen in serosae and can readily disseminate through the meningeal space; necrosis is usually more prominent in the meninges than in other organs. The infection can be congenital and it is usually found in young animals.

In cattle, the route of entry for M. bovis is usually the respiratory (by aerosols) and digestive (by ingestion of contaminated food, swallowed bronchial Mycobacterium containing exudate) systems; lesions are localized in the mesenteric lymph nodes. Cutaneous, congenital and genital are uncommon routes of entry for Mycobacterium sp.
In areas where the disease is common, up to 0.5% of the infection in newborn calves occurs through the umbilicus. As uterine tuberculosis is more common in cows than in any other species, congenital tuberculosis is very common in calves. Other than congenital infections, respiratory and digestive are the main routes in young animals. Abattoir surveys show that the prevalence of tuberculosis lesions in the nervous system is 0.35% to 0.53% while prevalence in other organs is as follows: lungs, 60.9%; liver 30.7%, peritoneum 24%, kidney 6%, udder 4.6%, reproductive system 7.5%, bones 3.8%.
Tuberculosis affecting the nervous system can have four presentations: (i) meningeal, (ii) isolated tubercles in the brain, (iii) spinal cord compression due to tuberculous osteomyelitis of the vertebral bodies, and (iv) tuberculous neuritis due to extension of lesions from other organs. The meningeal presentation is the most common. The clinical signs can be insidious or abrupt in onset and include fever, incoordination, apathy, seizures, blindness and opisthotonus. Gross lesions are usually marked. There is opacity of leptomeninges, and accumulation of fibrin-like exudate in the sulci, along the blood vessels.
AFIP Diagnosis: Cerebellum: Meningitis, granulomatous, diffuse, severe, with necrotizing vasculitis, mineralization, thrombosis, caseous necrosis, and multifocal granulomatous encephalitis, Holstein Friesian, bovine.
Conference Note: Mycobacterial infections are caused by bacteria belonging to the family Mycobacteriaceae, order Actinomycetales. Mycobacterium sp. are aerobic, weakly gram-positive, non-spore forming, non-motile bacilli with wide variations in host affinity. Mycobacteria stain with carbol dyes and resist subsequent decolorization with inorganic acids. This characteristic which is due to the spatial arrangement of mycolic acids within the cell wall makes them acid fast.
The ability of mycobacteria to survive and multiply within macrophages determines whether disease will occur within the host. Mycobacteria sp. utilize several virulence factors including cord factor or trehalose dimycolate, surface glycolipid, sulfatides, lipoarabinomannan, heteropolysaccharide, heat shock protein, complement, and tubuloprotein. The types of immune responses that are critical in responding to mycobacterial infection are cell-mediated immunity and the delayed hypersensitivity response.
The species causing "classic" tuberculosis are termed the M. tuberculosis complex (MTC) and include M. bovis, M. tuberculosis, M. africanum (rare cause of human TB in Africa), and M. microti (a rodent pathogen that has been reported to infect cats). Those species grouped together causing the syndrome of M. avium complex (MAC), sometimes referred to as "avian mycobacteriosis", include Mycobacterium avium-intracellulare and M. avium spp. paratuberculosis. The latter, which is the cause of Johne's disease in ruminants (ruminant paratuberculosis), can also infect monogastric animals.
Another separate group of myocobacterial infections is caused by M. leprae and the disease is termed leprosy or Hansen's disease. Feline and murine leprosy is caused by M. lepraemurium. The final group, atypical mycobacteriosis, can be described as localized opportunistic skin and subcutaneous infections caused by saprophytic and rapidly growing mycobacteria, e.g. M. fortuitum, M. chelonae, etc. Ziehl Neelsen acid fast stains provided by the contributor demonstrated small numbers of acid fast bacilli.
Contributor: Universidade Federal de Santa Maria, Departamento de Patologia, 97119-900, Santa Maria, RS, Brazil.
1. Andrade GB, Riet-Correa F, Mielke PV, Méndez MC, Shild AL: Estudo histológico e isolamento de micobactérias de lesões similares à tuberculose em bovinos no Rio Grande do Sul. Pesq. Vet Bras :81-86, 1991
2. Correa, CNM, Correa WM, Spago N, Matsumoto T: Tuberculose nervosa em vaca leiteira. Arq Esc Vet UFMG 32(2):265-269, 1980
3. Dahme E. Nervensystem Besonderer Formen bakterieller Infektionen. In: Grundriß der speziellen pathologischen Anatomie der Haustiere, eds. Dahme E, Weiss E, 4 ed., p. 535. Ferdinand Enke, Stuttgart, Germany, 1988
4. Dungworth DL: The Respiratory System. In: Pathology of domestic animals, vol 2, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp. 539- 699. Academic Press San Diego, 1993
5. Francis J: Bovine tuberculosis including a contrast with human tuberculosis. pp. 63-125. Stamples Press, New York, NY, 1947
6. Frauchingen E, Hofmann W III: Die Erkrankungen der Hüllen des Zentralnervensystems. In: Die Nervenkrankheiten des Rindes, Frauchinger E, Hofmann W, pp.217-247. Hans Huber, Bern, Switzerland, 1941
7. Guedes RMC, Nogueira RHG, Facury Filho EJ, Lago L A: Meningite tuberculosa bovina. Arq Bras Med Vet Zootec 49(1):131-135, 1997
8. Kantor IN: Regional and Country status reports. The Americas, In: Mycobacterium bovis infection in animals and humans, eds. Thoen CO, Steele JH, pp. 166- 202. Iowa State University, Ames, Iowa, 1995
9. Palaske G: Ablauf und pathologische Anatomie der Tuberkulose der verschiedenen Tierarten im besonderen. In: Pathologische Anatomie und Pathogenese der spontanen Tuberkulose der Tiere. ed. Pallaske G, p 54-99. Gustav Fischer, Stuttgart, 1961
10. Thoen C O, Himes E M: Mycobacterium. In: Pathogenesis of Bacterial Infections in Animals, pp. 26-37, Iowa State University Press, Ames, Iowa, 1986
11. Cotran RS, Kumar V, Collins T: Robbin's Pathologic Basis of Disease, 6th ed., pp.332-351. W.B. Saunders, Philadelphia, PA, 1999
Case II - 99-4943 (AFIP 2694953 )
Signalment: Tissues is from a 10-year-old, castrated male, Thoroughbred horse.
History: Owner reported horse had a swollen tongue for a period of 2 weeks. Swelling continued for an additional 2 weeks, at which time a biopsy was taken from the cranial portion.
Gross Pathology: A formalin-fixed, 1 x 1 x 2 cm piece of tongue was received.
Contributor's Diagnosis and Comments: Glossitis, granulomatous and eosinophilic, multifocal, with intralesional sarcocysts.

The most significant changes are eosinophilic granulomas, accompanying fibrosis, myofiber loss, and intramyofiber sarcocysts. Several granulomas center on sarcocyst capsular fragments or degenerative bradyzoites. Sarcocyst capsules have radial striations and are approximately 2 mm thick.1 The sarcocysts are presumptively identified as Sarcocystis fayeri, based on morphology and geographic location (S. fayeri is the only species of sarcocyst reported in equine muscle in the US).1,2 Sarcocystis sp. are obligate, coccidian parasites that undergo asexual reproduction in the vascular endothelium and myocytes of herbivorous intermediate hosts. Horses become infected by consuming feed contaminated with sporocysts from definitive hosts (dogs). Sarcocystis sp. infection rarely results in myositis, although cases similar to this one have been sporadically reported.2
AFIP Diagnosis: Tongue: Granulomas, eosinophilic, multiple, with myodegeneration, necrosis, regeneration, and intralesional and extralesional protozoa, Thoroughbred, equine, etiology consistent with Sarcocystis sp.
Conference Note: Conference participants unanimously agreed with the diagnosis of Sarcocystis myositis. Over 90 species of Sarcocystis have been recognized in mammals, birds, and reptiles, and at least 14 of these are regularly found in striated muscle or the intestine (as a form of alimentary disease caused by the sexual stages in the definitive, carnivorous host) of domestic animals. Often clinical disease does not occur. The severity of clinical signs varies with the species of parasite, the age of the infected animal, and the number of sporocysts ingested.
All Sarcocystis species have an obligatory two-host life cycle. Definitive hosts are carnivores, which are usually clinically unaffected. They prey on the herbivorous intermediate hosts. Upon being ingested by carnivores and released from mature cysts, zoites invade the intestinal epithelium and develop into gamonts. Fertilization occurs, followed by the formation of oocysts, which sporulate within the carnivore's intestine. Infective oocysts are shed in the feces. Susceptible herbivores then ingest oocysts or sporocysts, and sporozoites are released in the intestine and migrate into arterioles, where first generation merogony occurs in endothelial cells. Merozoites released from meronts undergo second generation merogony in capillary endothelium throughout the body. Upon subsequent liberation, merozoites enter circulating mononuclear cells and undergo endodyogeny (third generation merogony). Finally, zoites from second and third generation meronts enter the heart, skeletal muscle, or neural tissue (varies with species) and develop into immature noninfective sarcocysts containing unicellular metrocytes. Metrocytes produce bradyzoites that are infective for the definitive host, and whose presence characterizes a mature sarcocyst.
Contributor: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040.
1. Cawthorn RJ, Clark M, Hudson R, Friesen D: Histological and ultrastructural appearance of severe Sarcocystis fayeri infection in a malnourished horse. J Vet Diagn Invest 2:342-245, 1990
2. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues, 2nd ed., pp. 41-47. Armed Forces Institute of Pathology, Washington, DC, 1998
3. Hulland TJ: Muscles and Tendons. In: Pathology of Domestic Animals, vol. 1, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp. 257-260. Academic Press, San Diego, CA, 1993
4. Traub-Dargatz JL, Schlipf JW Jr., Granstrom DE, Ingram JT, Shelton GD, Getzy DM, Lappin MR, Baker DC: Multifocal myositis associated with Sarcocystis sp. in a horse. JAVMA 205:1574-1576, 1994
Case III - N 358/94 (AFIP 2506092)
Signalment: 15-month-old female red deer (Cervus elaphus).
History: This hind was from a herd of 50 farmed deer. It was one of four similarly affected deer from this herd. All four deer developed ill-thrift and intermittent diarrhea while remaining bright and alert. This hind was euthanized.
Gross Pathology: Numerous pinhead sized white foci were scattered throughout the liver. The wall of the small intestine was thickened particularly in the area of the terminal ileum, and the luminal surface of both large and small intestine exhibited transversely arranged rugae. The mesenteric lymph nodes were marked enlarged and there was an associated lymphangitis.
Laboratory Results: Numerous acid-fast bacilli were seen in smears made from material taken from the mucosa of the large and small intestine and from the lymph nodes. Mycobacterium paratuberculosis was isolated on cultural examination of the mucosa.
Contributor's Diagnoses and Comments: Granulomatous hepatitis, enteritis and lymphadenitis associated with M. paratuberculosis infection.
There are numerous small granulomatous foci randomly scattered throughout the liver. These granulomas are composed of foamy macrophages admixed with occasional lymphocytes. Large numbers of acid-fast organisms were seen to be associated with these granulomas on examination of Ziehl-Neelsen-stained sections (see transparency). Histopathological examinations of other organs confirmed the presence of a granulomatous lymphadenitis and enteritis also associated with the presence of acid-fast organisms. Johne's disease is uncommon in cattle and sheep in Ireland. However, it has emerged as a significant condition in farmed red deer. Onset of clinical disease may occur in animals as young as one-year- old. In addition, lesions may occur in organs such as the lung and the liver as well as in the intestine and its associated lymph nodes.
AFIP Diagnosis: Liver: Hepatitis, granulomatous, portal and multifocal, moderate, with intrahistiocytic bacteria, red deer (Cervus elaphus), cervid.
Conference Note: Mycobacterium spp. are aerobic, facultative intracellular, weakly gram-positive, non-spore forming, non-motile bacilli with wide variation in host affinity. The bacterial cell wall is composed of complex lipids including glycolipids, lipopolysaccharides, lipoproteins, and waxes. Mycolic acid is the lipid that confers the acid-fast property.
Mycobacterium avium, ssp. paratuberculosis causes chronic granulomatous ileitis/colitis, with associated villus atrophy, and regional lymphadenitis (Johne's Disease) in ruminants.
Paratuberculosis is a disease of major economic importance to the dairy and beef cattle industries. In addition to cattle, natural disease occurs in sheep, goats, and llamas, and has been described in white-tailed deer, red deer, bighorn sheep, Rocky Mountain goats, and other wild ruminants. Some strains are specific for goats and non-pathogenic to cattle.
Paratuberculosis occurs worldwide and appears to be spreading insidiously. Channel Island breeds (Jersey and Guernsey) and beef shorthorn cattle and some breeds of sheep may be more susceptible. Since many infected cows appear healthy, and there is no reliable antemortem detection method, the disease is easily spread. Although only 1-2% of a herd may be clinically ill, 40-100% may be infected. Cattle of an involved herd are divided into four categories: 1) infected with clinical signs; 2) asymptomatic shedders; 3) asymptomatic nonshedders; 4) non-infected.
The organism survives in the environment for 6-9 weeks. Calves are usually infected by 3-6 months of age by ingesting contaminated feces. In older animals macrophages can restrict intracellular growth of bacteria, although lysis does not occur, conferring age-dependent resistance to clinical disease. Organisms cross the intestinal mucosa and enter macrophages in Peyer's patches and local lymph nodes. Gross and histologic lesions are usually confined to the ileum, large intestine and draining lymph nodes, but the infection is systemic. In fulminating infections, bacteremia may occur. Bacteria are shed mainly in feces, although organisms may be excreted in milk, semen, urine and uterine secretions. Bovine fetuses may be infected as early as the second month of gestation. Similarly, embryos within the uterus of superovulated cows can be infected, as the bacteria have been shown to adhere to ova. This infection then may be spread to surrogate cows.
M. avium ssp. paratuberculosis has been hypothesized to be the cause of Crohn's disease in man. Crohn's disease is a granulomatous condition affecting the lower ileum and often the colon. Although lesions are somewhat similar, convincing evidence of a causal association has not been produced.
Contributor: Dept. of Veterinary Pathology, Faculty of Vet. Medicine, University College Dublin, Shelbourne Road, Ballsbridge, Dublin 4, Ireland.
1. Barker IK, Van Dreumel AA: The Alimentary System. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N. 4th ed., vol 2, pp. 247-252. Academic Press, New York, NY, 1993
2. Power SB, Haagsma J, Smyth DP: Paratuberculosis in farmed red deer (Cervus elaphus) in Ireland. Vet Rec 132(9):213-6, 1993
3. Van Kruiningen HJ: Lack of support for a common etiology in Johne's disease of animals and Crohn's disease in humans. Inflamm Bowel Dis 5(3):183-91, 1999
4. Williams ES, Snyder SP, Martin KL: Pathology of spontaneous and experimental infection of North American wild ruminants with Mycobacterium paratuberculosis. Vet Path 20:274-291, 1983
Case IV - 99-231 (AFIP 2694980)
Signalment: 4-year-old, male, black Labrador, dog.
History: Dog originally from California. Presented to the veterinary teaching hospital for epistaxis and respiratory distress. While hospitalized the dog went into respiratory arrest and died.
Gross Pathology: Numerous, 10 cm long x 0.3-cm diameter, white worms were in the right ventricle and caudal vena cava. The intimal surface of the proximal pulmonary artery was irregular with a granular appearance. The heart was not enlarged. All lung lobes were mottled light to dark red.
Laboratory Results: A moderate leukocytosis, characterized by neutrophilia with a left shift, monocytosis and eosinophilia, was present. Microfilaria morphologically consistent with Dirofilaria immitis were seen on blood smears.

Contributor's Diagnoses and Comments:
1. Angiotrophic lymphoma, lung
2. Microfilaremia

The most significant change is the accumulation of neoplastic round cells within the pulmonary vasculature. Neoplastic cells widely extend into vessel walls, and occasional vessels are completely occluded. Intravascular aggregates of fibrin are also evident, both associated and unassociated with neoplastic cells. Immunohistochemical stains using a polyclonal primary antibody directed against the T-lymphocyte marker CD3 were performed on replicate sections and CD3 antigen was identified in neoplastic cells. Small numbers of microfilaria are among erythrocytes within blood vessels, including septal capillaries.
Angiotrophic lymphoma, also known as malignant angioendotheliomatosis, is a rare neoplastic disorder described in humans, dogs, and cats. Using immunoglobulin markers, neoplastic cells have been identified as either of B- or T- cell origin. The proliferation of neoplastic cells within the vasculature results in thrombosis and tissue infarction, which often are responsible for presenting clinical signs. The central nervous system and skin are common sites in which neoplastic cells are initially identified, although any tissue can be affected.
In the present case, neoplastic cells were identified in kidney, lung, pituitary gland, spleen, esophagus, liver, nasal mucosa, prostate, multiple lymph nodes, intestine, thyroid gland, adrenal gland, pancreas, and brain. Skin was not examined. In affected animals, the clinical course is typically rapid, often resulting in death (or euthanasia) within several weeks of the initial diagnosis. Antemortem diagnosis is difficult, as no distinct clinicopathologic or radiographic findings exist. Of special note is that neoplastic cells do not circulate and consequently are not evident on blood smears; hence biopsy of affected tissue is frequently necessary to establish a diagnosis. In this case, the clinical suspicion was that the dog died from pulmonary thromboembolism secondary to dirofilariasis; the presence of the angiotrophic lymphoma was unanticipated.
AFIP Diagnosis:
1. Lung: Malignant lymphoma, intravascular, black Labrador, canine.
2. Lung: Thrombosis, multifocal.
3. Lung: Arteriosclerosis, multifocal.
4. Lung: Microfilaria, intravascular, numerous.
Conference Note: The term "angioendotheliomatosis" has caused confusion as it refers to two very different vascular lesions. One originally called "malignant angioendotheliomatosis" has been found to be intravascular lymphoma. The other, "reactive angioendotheliomatosis" has been reported in cats and humans and is characterized by proliferation of endothelium and pericytes. However, the feline and the human diseases are radically different: the former is fatal and the latter is benign.

Conference participants agreed that there is significant vascular sclerosis but could not determine whether this lesion was the result of heartworm disease or intravascular lymphoma. Both have been reported to cause this lesion. Immunohistochemical staining for CD-3 and CD-79 performed at the Armed Forces Institute of Pathology, failed to work on two attempts. Generally canine intravascular lymphoma is of a T-cell origin, whereas most cases of intravascular lymphoma in humans are B-cell lymphomas.
Contributor: Department of Veterinary Microbiology and Pathology, Washington State, University, Pullman, WA 99164-7040
1. Fuji R, Freels K, Summers B: Systemic reactive angioendotheliomatosis in cats: Two cases and a review of the literature. ABSTRACT Vet Pathol 35(5):420, 1998
2. Kilrain CG, Saik JE, and Jeglum KA: Malignant angioendotheliomatosis with retinal detachments in a dog. JAVMA 204:918-921, 1994
3. LaPointe JM, Higgins RJ, Kortz GD, Bailey CS, Moore PF: Intravascular malignant T-cell lymphoma (malignant angioendotheliomatosis) in a cat. Vet Pathol 34:247-250, 1997
4. Perniciaro C, Winkelmann RK, Daoud MS, Su WP: Malignant angioendotheliomatosis is an angiotropic intravascular lymphoma. Immunohistochemical, ultrastructural, and molecular genetics studies. Am Journal Dermatopathol 17:242-248, 1995
J Scot Estep, DVM
Captain, United States Army
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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