AFIP Wednesday Slide Conference - No. 6
13 October 1999

Conference Moderator:
Dr. Timothy O'Neill, Diplomate, ACVP
Biomedical Research Consultants Inc.
Middletown, MD 21769-6704
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Case I - 2058 (AFIP 2694987)
Signalment: A four-month-old, female, Suffolk lamb (Ovis ovis).
History: The lamb was at pasture, doing well, when she suddenly developed anorexia and ataxia, followed by recumbency. The lamb died within three days of first showing clinical signs. The owner gave no treatment. A veterinarian, who was called to examine the animal shortly before it died, suspected polioencephalomalacia. Four other lambs in the group were unaffected.
Gross Pathology: There were no significant findings on the gross necropsy carried out by the practitioner. Fresh and fixed tissues were submitted for further study.
Laboratory Results:
Bacteriology: Listeria monocytogenes was cultured from the brain in heavy growth. Light to heavy growth of E. coli was cultured from the lung and spleen.
Toxicology: Kidney and liver samples were negative for lead and cadmium. Liver analyses for Se, Cu, Zn, Fe, Mn, were within adequate levels.

Contributor's Diagnosis and Comments: Meningoencephalitis (brain stem and cerebellum), subacute, severe, mixed cellular to suppurative, with mononuclear perivascular cuffing, neuropil edema and microabscessation.
Significant pathological changes in submitted tissues were limited to the cerebellum and brain stem, notably the medulla. These are characterized by heavy mononuclear perivascular inflammatory infiltrates consisting of histiocytes, lymphocytes and plasma cells; a few neutrophils are also seen. Edema of the affected areas of the neuropil is prominent and, particularly in sagittal brain sections, there are multifocally extensive inflammatory infiltrates with areas of gliosis, microabscessation and focal neuronal necrosis that extend into the cerebellar corpus medullare. Depending upon which sections are examined, meninges over affected brain areas show a variable inflammatory infiltrate, from mild to focally severe.

In this case, small Gram positive bacilli or coccobacilli resembling L. monocytogenes were observed most readily within dense inflammatory cell foci, some of these being microabscesses. The organisms demonstrated pleomorphism, appearing as small rods or as coccobacilli. The organisms were seen in small, irregular clumps or occasionally in short chains and appear to be extracellular. Less readily defined and stained individual organisms were also seen within the background that may represent an intracellular, cytoplasmic, location.

Sagittal and transverse sections of brain are included. Many of the sagittal sections show extensive lesions. Transverse sections tend to show affected areas that are more focal or patchy. The relatively regional area of affected brain tissue within the brain stem (pons, medulla) and cerebellum emphasizes the importance of receiving these areas for both culture and for histopathology from field practitioners seeking a diagnosis. Mild autolysis is present.
L. monocytogenes is a small Gram positive, facultative intracellular bacillus that may cause disease in most animal species, including man (1). Listeriosis in sheep, as in most other affected species, occurs in 3 distinct syndromes which seldom overlap (1, 2). These are encephalitis; systemic infections; and abortion. Less commonly, L. monocytogenes is a cause of endocarditis and purulent lesions in other organs and tissues (1). Clinical listeriosis is evidently rare in horses, pigs, dogs, and cats (3). The organism is ubiquitous in nature and can be recovered from soil, vegetation, dairy products, animal feces, and sometimes the oropharynx and tissues of healthy animals (1, 2, 3).
The disease tends to be seasonal, with clinical cases reported more commonly in winter or early spring (3); the case reported here occurred in late May. There is a strong association between listeriosis and animals fed silage rather than hay. This young lamb was on pasture grass.
Clinical signs of "circling disease" were not described in this animal, although it was reported as wobbly or ataxic prior to recumbency. The pathogenesis of listerial encephalitis is still only partially understood (2). In the encephalitic form, the organism is thought to invade the brain via the cranial nerves (2, 3). Trigeminal neuritis has been reported in 16 of 17 sheep that were spontaneously affected by listerial encephalitis; the distribution of lesions in sheep brains also suggested movement along axon fiber tracts (4). The bulk of evidence is against the hematogenous route (2). It is not clear how L. monocytogenes breaches the oral mucosal epithelium, but it has been suggested that it may penetrate the dental pulp when sheep are cutting or losing teeth (3). It is interesting that in the ruminant brain and occasionally in the human brain, the lesions caused by L. monocytogenes are regionally disseminated small foci of inflammatory cells, whereas those caused by other pyogenic infections usually produce one or several large abscesses (4). The organism has been demonstrated in myelinated axons of the trigeminal nerve and fiber tracts in the brain stem, and within the cytoplasm of medullary neurons (2,4); in foci of inflammation it has been reported as more commonly seen in neutrophils than in macrophages (4).

The potential for L. monocytogenes to demonstrate pleomorphism has been well recorded elsewhere, including its ability, in Gram stains of clinical specimens, to resemble streptococci, to which it is genetically related (5). Variations in the appearance of L. monocytogenes grown on culture plates and those identified in clinical specimens is an interesting point (5), and any comments by conference participants is welcomed.
Progress has been made in identifying virulence factors in L. monocytogenes in mice and tissue culture cells, and presumably these are general principles which influence its virulence for sheep as well (3). The organism must invade a cell, including epithelial cells and phagocytes; it multiplies within cell cytoplasm where, in a poorly understood manner, it appears to be propelled peripherally. Projections of the infected cell membrane then invaginate into adjacent cells, and transmit the listeriae. As a result, L. monocytogenes is able to multiply and spread without direct exposure to the cells and soluble factors of the extracellular environment (3).
Listeria monocytogenes is considered a zoonotic agent. Infection may be transmitted to humans directly from infected meat (e.g., hot dogs) or milk, or indirectly from infected cheese made from unpasteurized milk (2,3).
AFIP Diagnosis: Brain stem and cerebellum: Meningoencephalitis, subacute, multifocal, moderate, with microabscesses, Suffolk lamb (Ovis ovis), ovine.
Conference Note: Conference participants agreed with the contributor's description of the lesions and morphologic diagnosis. The contributor has provided an excellent review of listeriosis.

Contributor: Animal Health Centre, 1767 Angus Campbell Rd, Abbotsford, British Columbia, Canada V3G 2M3.
1. Jones CT, Hunt RD, King NW: Veterinary Pathology, 6th ed, pp. 461-462, Williams and Wilkins, Philadelphia, PA, 1997
2. Jubb KVF, Huxtable CR: The nervous System. In: Pathology of Domestic Animals, 4th ed., vol 1, eds. Jubb KVF, Kennedy PC, Palmer N. pp. 393-397, Academic Press Inc., NY, 1993
3. Czuprynski CJ: Listeria. In: Pathogenesis of Bacterial Infections in Animals, eds. Gyles CL, Thoen CO, 2nd ed., pp. 70-79, Iowa State Univ Press, Ames, Iowa, 1993
4. Charlton KM, Garcia MM: Spontaneous listeric encephalitis and neuritis in sheep. Light microscopic studies. Vet Pathol 14:297-313, 1977.
5. Mielke MEA, Thomas KH, Unger M: Listeriosis In: Pathology of Infectious Diseases, vol 1 eds. Connor DH, Chandler FW, Baird JK, Schwartz DA, Lack EE, Utz JP, pp. 621-633, Appleton & Lange, Stanford, Connecticut, 1997
Case II - B97-8011, 97-14-4 or 97-14-8 or 97-14-9 or 97-15-1 (AFIP 2683969)
Signalment: A four-month-old, male, Hanford-HA minipig (Sus scrofa domesticus)
History: The sections are from the hearts of two untreated positive control Hanford minipigs used in a research study to investigate reperfusion injury following surgical occlusion of the left anterior descending (LAD) artery. One week prior to necropsy, the LAD had been occluded for 45-60 minutes followed by reperfusion, creating a myocardial infarction. As part of the procedure, the left ventricle and great vessels were instrumented. Additionally, fluorescent microspheres were injected (peripherally) during the study period.
Contributor's Diagnoses and Comments:
1. Severe sub-acute locally extensive intramural myocardial infarction with granulation tissue.
2. Mild to moderate sub-acute diffuse reactive visceral pericarditis.
3. Intravascular microspheres (not present in all sections).
Most sections provided are full thickness through the left ventricular free wall. The most prominent lesion is locally extensive intramural sub-acute myocardial infarction, characterized by complete loss of myocardial tissue with replacement by granulation tissue. In some sections, islands of myocardium are present within the damaged region. The interface between infarcted and intact tissue is usually abrupt, but there are some examples of an infiltrating pattern. The amount of edema associated with the granulation tissue varies among the sections. Neutrophils are present, both free in the granulation tissue and marginated in smaller blood vessels. Degenerating tissue, characterized by nuclear debris (karyorrhexis), is scattered throughout the affected tissue.
Generally, the subendocardial and subepicardial regions contain relatively intact cardiac muscle but some myocardial tissue contains rarefied, vacuolated cytoplasm. Intact Purkinje fibers are present in some sections. The visceral pericardium is mildly to severely reactive, typified by granulation tissue with a variety of inflammatory cells, predominately neutrophils. Multinucleate giant cells are present in a few sections. Some sections lack the pericardial surface. Microspheres (15-17µ in diameter) are present within small vessels in intact myocardium in many sections.
AFIP Diagnosis: Heart, left ventricle: Cardiomyocyte loss, focally extensive, with granulation tissue, multifocal mild lymphocytic, histiocytic and neutrophilic inflammation and focal chronic proliferative pericarditis (infarct), Hanford-HA minipig, porcine.
Conference Note: The differential diagnosis discussed in conference included ischemic injury, vitamin E/ selenium deficiency, and viral infection. However, the discrete loss of myofibers with replacement by granulation tissue and mild inflammation were considered to be most consistent with a subacute to chronic ischemic insult (infarct).
Ischemia and reperfusion both damage cardiomyocytes. The severity of injury from infarction is directly related to the duration of occlusion, location of occlusion and metabolic needs of the myocardium. Ischemia results in depletion of ATP (within seconds), loss of contractility (<2 minutes), irreversible cell injury (20-40 minutes), and microvascular injury (>1 hour). Reperfusion can salvage sublethally damaged cardiomyocytes, but can also lead to further injury. Reperfusion increases the generation of oxygen free radicals, supporting the release of cytokines from damaged cells and the recruitment of inflammatory cells that cause further injury.
Histologic staging of infarction follows a distinct pattern based on the time after injury. First signs of coagulation necrosis occur between 4 and 12 hours. Within 1-3 days, there is continued coagulation necrosis, loss of cross striation and infiltration of the interstitium by neutrophils. From days 3-7, there is degeneration of neutrophils and disintegration of myofibers with peripheral phagocytosis by macrophages. After 7-10 days, there is well-developed phagocytosis and formation of granulation tissue.

After two weeks, there is a gradual decrease in cellularity and deposition of collagen, eventually resulting in a dense scar. Based on these characteristics, the histologic features present in this case are in close agreement with the clinical history of a one-week-old ischemic injury.
Contributor: The Procter & Gamble Company, Miami Valley Laboratories, PO Box 398707, Cincinnati, Ohio.
Schoen FJ: The Heart. In: The Pathologic Basis of Disease, eds. Cotran RS, Kumar V, Collins T, 6th ed., pp. 550-563. WB Saunders Company, Philadelphia, PA, 1999
Case III - A41177 (AFIP 2694681)
Signalment: Six-month-old pig (breed and sex unknown).
History: This pig was presented for slaughter at a federally inspected establishment. The pig was not identified as abnormal during antemortem inspection.
Gross Pathology: There was hepatomegaly and splenomegaly with accumulation of yellow material with a lipid-like texture. The lungs and parietal pleura were discolored (yellow) and there was a mottled appearance of cut surfaces of thoracic and lumbar lymph nodes. Mesenteric lymph nodes were enlarged and cut surfaces were homogenously yellow-orange with a soft texture. The jejunal mucosa contained yellow plaques. Adipose tissue in skeletal muscle and heart appeared normal.
Contributor's Diagnoses and Comments:
1. Lymph node, Spleen, Liver, Lung: Histiocytosis, diffuse, marked, with intrahistiocytic vacuoles.
2. Liver: Hepatocellular lipidosis, diffuse, marked.
3. Jejunum: Histiocytosis, diffuse, marked with Touton-type giant cells, foam cell thrombi, and atheromatous plaques.

Frozen sections revealed that vacuoles in macrophages and hepatocytes contained lipid. Special stains for bacteria and fungi revealed no etiologic agents. One possible etiology for these lesions would be hyperlipidemia secondary to an inherited defect in lipid metabolism, perhaps combined with a high fat diet. Inbred swine are a key animal model for study of human familial hypercholesterolemia, but lesions of this severity are not generally seen in 6- month-old animals.
Another consideration would be an inherited lipid storage disease. Two lysosomal storage diseases have been suggested to occur in pigs. There is a well-defined GM2 gangliosidosis of Yorkshire pigs that results in primarily neuronal storage. There also is a single report of a less well-defined disease thought to be similar to glucocerebrosidosis in which there was vacuolization of cells in the liver, spleen, and in other visceral organs.
AFIP Diagnoses:
1. Small intestine: Histiocytosis, diffuse, severe, with lipid-type cytoplasmic vacuolation, histiocytic lymphatic emboli and Touton giant cells, breed unknown, porcine.
2. Spleen: Histiocytosis, multifocal, moderate, with lipid-type cytoplasmic vacuolation, and multifocal reticuloendothelial cell hyperplasia.
3. Lung: Histiocytosis, interstitial, diffuse, moderate, with lipid-type cytoplasmic vacuolation, histiocytic lymphatic emboli and mild subacute interstitial pneumonia.
4. Liver: Histiocytosis, centrilobular, bridging, portal, and sinusoidal, mild to moderate with lipid-type vacuolation, and mild to moderate diffuse lipid-type hepatocellular cytoplasmic vacuolation.
5. Lymph node: Histiocytosis, diffuse, severe, with lipid-type cytoplasmic vacuolation and scattered eosinophils.
Conference Note: The conference participants essentially agreed with the contributor's diagnosis. Differential diagnosis discussed in conference included fungal infections, mycobacteriosis, storage diseases (ceroid-lipofuscinosis and GM2 gangliosidosis) and hypercholesterolemia/ hyperlipemia. Clinical history, blood lipid profile, knowledge of the breed and diet, evaluation of related pigs, and electron microscopy could provide additional information to help differentiate storage diseases from hypercholesterolemia/hyperlipemia.
Contributor: USDA FSIS OPHS Eastern Laboratory, PO Box 6085, Russell Research Center, 950 College Station Road, Athens, GA, 30604
1. Attie AD: The spontaneously hypercholesterolemic pig as an animal model for human atherosclerosis. ILAR News 30:5-12, 1988
2. Jolly RD, Walkley SU: Lysosomal storage diseases of animals: an essay in comparative pathology. Vet Pathol 34:527-548, 1997
3. Kosanke SD, Pierce KR, Bay WW: Clinical and biochemical abnormalities in porcine GM2 gangliosidosis. Vet Pathol 15:685-699, 1978
4. Kosanke SD, Pierce KR, Read WK: Morphogenesis of light and electron microscopic lesions in porcine GM2 gangliosidosis. Vet Pathol 16:6-17, 1979
5. Pierce, KR, Kosanke SD, Bay WW, Bridges CH: GM2 Gangliosidosis, Model No. 104. In: Handbook: Animal Models of Human Disease, Fasc. 6, Eds. TC Jones, DB Hackel, G Migaki. Registry of Comparative Pathology, AFIP Washington, DC, 1977
6. Sandison AT, Anderson LJ: Histiocytosis in two pigs and a cow: conditions resembling lipid storage disorders in man. J Pathol 100:207-210, 1970
Case IV - 96-636 (AFIP 2694731)
Signalment: One-year-old, breed not specified, male guinea pig (Carva cobaya)
History: Found dead in an animal nursery.
Gross Pathology: Consolidation of all pulmonary lobes, marked splenic hyperplasia, moderate lipidosis of the liver, multiple hemorrhages of the mucous membranes of stomach, colon and rectum.
Laboratory Results: Lung: E. coli, Klebsiella oxytoca, Pasteurella haemolytica, Lactobaccillus sp., Moraxella sp.

Contributor's Diagnoses and Comments:
1. Lung: pneumonia, interstitial, diffuse, severe with numerous macrophages within the alveolar lumina and basophilic intranuclear inclusion bodies.
2. Lung vessels: perivascular edema, severe, acute thrombosis of small vessels and basophilic intranuclear inclusion bodies in endothelial cells.
3. (Not submitted) Liver: moderate lipidosis, basophilic intranuclear inclusion bodies in hepatocytes and endothelial cells.
4. Spleen: basophilic intranuclear inclusion bodies in unidentified cells.
Etiology: consistent with adenovirus infection.
The diagnosis of adenovirus-infection was established by electron-miroscopic investigation. Within the nuclei of hepatic endothelial cells, numerous round virus particles were identified. They measure approximately 90nm in diameter and some have an electron-dense core while others have an electron-lucent core. The size and appearance of the viral particles are characteristic for adenovirus. The first reports on a spontaneous respiratory disease in guinea pigs caused by adenovirus (Namann et al., 1981; Brennecke et al., 1983) gave reason for an experimental proof of the infection (Kaup et al., 1984; Kunsty et al., 1984).
Case 6-4. Note multiple electron dense viral particles and other immature membrane bound particles which contain developing central nucleoids.
The case presented was observed 2 years after a first case of adenovirus-infection in a guinea pig, equally examined by EM. Unfortunately, culture of the virus was not attempted; hence it is not feasible to confirm the diagnosis. With regard to the literature cited, there is not much doubt about the nature of the virus. Both cases are described in detail in the article cited in the references below.
AFIP Diagnosis: Lung: Pneumonia, interstitial, subacute, diffuse, moderate, with multifocal fibrin thrombi and intrahistiocytic and endothelial basophilic and eosinophilic intranuclear inclusion bodies, guinea pig (Carva cobaya), rodent.
Conference Note: Although pneumonia is a common cause of death in guinea pigs, the cause is usually bacterial. Common etiologic agents include Bordetella bronchiseptica, Streptococcus pneumoniae, Streptococcus zooepidemicus, Klebsiella pneumoniae, and Pasteurella multocida. Relatively common causes of viral infections in guinea pigs include cytomegalovirus, lymphocytic choriomeningitis virus and a few enteric viruses. Adenoviral pneumonia was diagnosed and experimentally reproduced in the early 1980's but has been reported rarely. Adenoviruses cause natural respiratory disease in cattle, sheep, horses, quail, nonhuman primates, dogs, and man, and experimental disease in swine and mice.
Differential diagnosis considered for this case included cytomegalovirus and adenovirus. Both of these viruses produce a similar histologic appearance, with large intranuclear inclusions. Cytomegalovirus causes prominent cytomegaly and by electron microscopy, there are 100-150 nm diameter, hexagonal viral nucleocapsids within nuclei of infected cells. Electron microscopy of adenovirus infected cells demonstrates 70-90 nm virions that are sometimes arranged in paracrystaline arrays.

The bar in the submitted electronmicrograph equals 100nm. The size and morphology of the viral particles are consistent with an adenovirus. An immunohistochemical stain for adenovirus performed at the AFIP was positive.
Contributor: Institute of Veterinary Pathology, Veterinaerstr. 13, 80539 Muenchen, Germany.
1. Brennecke C H, Dreier TM, Stokes WS: Naturally occurring virus-associated respiratory disease in two guinea pigs. Vet Pathol 20:488-491, 1983
2. Breuer W, Haunichen T, Hermanns W: Adenovirus-induced pneumonia in two guinea pigs. Berl Munch Tieruerztl Wschr, 110:51-53, 1997
3. Feldman SH, Richardson JA, and Clubb FJ, Jr: Necrotizing viral bronchopneumonia in guinea pigs. Lab Anim Sci 40:82-83, 1990.
4. Harris IE, and Goydich W: Adenoviral bronchopneumonia of guinea pigs. Aust Vet J 62:317, 1985
5. Kaup FJ, Naumann S, Kaup FJ, Kraft V, Knocke KW: Adenovirus pneumonia in guinea pigs: an experimental reproduction of the disease. Lab Anim 18:55-60, 1984
6. Kunstyr I, Maess J, Naumann S, Kaup FJ, Kraft V, and Knocke KW: Adenovirus pneumonia in guinea pigs: an experimental reproduction of the disease. Lab Anim 18:55-60, 1984
7. Naumann S, Kunstyr I, Langer I, Maess J, Hoerning R: Lethal pneumonia in guinea pigs associated with a virus. Lab Anim 15:235-242, 1981
J Scot Estep, DVM
Captain, VC, USA
Registry of Veterinary Pathology*
Department of Veterinary Pathology
Armed Forces Institute of Pathology
(202)782-2615; DSN: 662-2615
* The American Veterinary Medical Association and the American College of Veterinary Pathologists are co-sponsors of the Registry of Veterinary Pathology. The C.L. Davis Foundation also provides substantial support for the Registry.
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