AFIP Wednesday Slide Conference - No. 7
20 October 1999

Conference Moderator:
COL Kelly Davis, Diplomate, ACVP
Pathology Division
U.S. Army Medical Research Institute of Infectious Disease
Ft. Detrick, Frederick, MD 21702-5011
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Case I 3G (AFIP 2678957)
Signalment: 5-year-old male rhesus monkey (Macaca mulatta).
History: Infected with simian immunodeficiency virus (SIV) one year prior to euthanasia. Weakness, paraparesis before euthanasia.
Gross Pathology: No visible lesions.
Contributor's Diagnoses and Comments:
1. Cauda equina, neuritis, suppurative and hemorrhage, multifocal, acute, moderate.
2. Cauda equina, meningitis, suppurative and hemorrhage, multifocal, acute, moderate.
3. Spleen and lymph node, lymphoid depletion, diffuse, chronic, severe.
4. Liver, hepatitis, multifocal to coalescing, chronic, ongoing, moderate with fibrosis and occasional intranuclear inclusions.

Occasional to numerous intranuclear inclusions were seen in macrophages in the meninges in the cauda equina. Polymerase chain reaction testing was performed on spinal cord tissue and was positive for cytomegalovirus (CMV). The inclusions in the liver were also typical of CMV. CMV is a common opportunistic infection in immunosuppressed macaques. This SIV-infected macaque was immunosuppressed as suggested by severe lymphoid depletion of the spleen and lymph nodes.
AFIP Diagnoses:
1. Cauda equina: Neuritis and meningitis, necrohemorrhagic, focally extensive, severe, with karyomegaly, cytomegaly and eosinophilic intranuclear inclusion bodies and rare intracytoplasmic inclusion bodies, rhesus monkey (Macaca mulatta), non-human primate, etiology consistent with cytomegalovirus.
2. Nerve roots and ganglia: Ganglioneuritis, subacute, multifocal, mild, with karyomegaly, cytomegaly and eosinophilic intranuclear inclusion bodies and rare intracytoplasmic inclusion bodies, etiology consistent with cytomegalovirus.
Conference Note: Cytomegaloviruses are host-specific members of the subfamily betaherpesvirinae within the herpesvirus family. Man, non-human primates and a variety of other animals are commonly infected. Generally, clinical disease is restricted to immunocompromised individuals. Latent infection is common in rhesus monkeys; immunosuppression often leads to clinical disease.
When clinical disease occurs, it tends to be disseminated. In macaques infected with SIV, CMV most commonly affects the lungs, lymph nodes, small intestine, liver and brain. Infection generally produces characteristic karyomegalic cells with prominent intranuclear inclusions often surrounded by a clear halo ("owl's eye cells"). Rarely, smaller granular intracytoplasmic inclusions may be found. Some sections contain cells with intracytoplasmic inclusions of this type. Some conference participants thought that these inclusions might be protozoal organisms.

Contributor: Division of Comparative Medicine, 459 Ross Building, Johns Hopkins University, 720 Rutland Avenue, Baltimore, MD 21205.
1. Baskin GB: Disseminated cytomegalovirus infection in immunodeficient rhesus monkeys. Am J Pathol, 129(2):345-352, 1987.
2. Kuhn EM, Stole K, Matz-Rensing K, Mach M, Stahl-Henning C, Hunsmann G, Kaup FJ: Immunohistochemical studies of productive rhesus cytomegalovirus infection in rhesus monkey (Macaca mulatta) infected with simian immunodeficiency virus. Vet Pathol 36(1):51-56, 1999
3. Murphy FA, Gibbs EBJ, Horzinek MC, Studdert MJ: Veterinary Virology, 3rd ed., pp. 301-325. Academic Press, San Diego, CA, 1999
Case II - 99-0581 (AFIP 2694784)
Signalment: Adult, domestic shorthair, intact, female, cat, Felis domesticus.
History: This cat was exposed by the oronasal route to 50,000 TCID50 of a non-plaque purified strain of Nipah virus originally isolated from a human who died following a febrile encephalitis. Six days post exposure the cat developed fever and increased respiratory rate. Respiratory signs progressed to severe dyspnea and the cat was euthanized on day 9.
Gross Pathology: At necropsy the lungs did not collapse on opening the thorax; the lobes were "wet" and had a diffuse blotchy hemorrhagic appearance. There was purplish-red consolidation of the right apical and intermediate lung lobes and sanguinous frothy fluid in the bronchi. The tracheal mucosa was hemorrhagic particularly at the thoracic inlet. Mediastinal edema was noted together with increased (15ml) sanguinous free pleural fluid. The carcass was dehydrated and slightly icteric.
Laboratory Results: Nipah virus was isolated from lung, tonsil, spleen, and urine. Positive reactions by immunoperoxidase testing using rabbit anti-Hendra virus antibody were identified in lung, meninges, lymphoid tissues and blood vessels (endothelium and muscle). Specifically, immunostaining was noted in pulmonary alveolar epithelium, epithelial syncytia, bronchiolar epithelium and bronchoalveolar debris.

Contributor's Diagnosis and Comments: Lung: Focal to diffuse acute alveolitis and vasculopathy with epithelial and endothelial syncytia, edema, caused by infection with Nipah virus.
Further histologic lesions included mild essentially non-suppurative meningitis; individual cell necrosis of lymphoid cells; generalized necrosis of lymphoid tissue (possibly a consequence of infarction); syncytia formation in lymphoid tissue, particularly in paracortical areas; and acute vasculitis with fibrinoid necrosis of vessel walls in various tissues. Observation of immunopositivity to anti-Hendra virus antibody in respiratory epithelium together with airway debris suggests that, in contrast to Hendra virus, Nipah virus may at least be transmitted by bronchial secretions.
Between September 1998 and April 1999 over two hundred cases of human febrile encephalitis (over 100 resulting in death) were reported to the Malaysian Ministry of Health. These cases occurred primarily in adult men with a history of close contact with swine. At the same time a poorly defined disease syndrome causing both illness (cough, dyspnea, tremors, irritability) and death was noted in pigs from the same regions. Nine cases of human illness also occurred in abattoir workers handling Malaysian pigs. Until March 1999 the human disease was attributed to Japanese Encephalitis when a novel paramyxovirus was isolated from one of the people who had died.
Initial characterization of the virus suggested that the most closely related known virus was Hendra virus. The novel virus, subsequently named Nipah virus, exhibited approximately 20% variation from Hendra virus at the nucleotide level and 10% variation at the amino acid level for the first 2 genes studied. Nipah virus was confirmed to be the etiologic agent of the disease syndrome present in Malaysian pigs and an extensive program of culling of pigs from infected properties was initiated.
Further investigations on field samples from Malaysia have indicated the host range for Nipah virus includes horses, cats, bats, and probably dogs, as well as pigs and people. There is an obvious parallel to Hendra virus in the developing understanding of the role of bats in the epidemiology of Nipah virus in Malaysia.
AFIP Diagnosis: Lung: Pneumonia, bronchointerstitial, necrotizing, fibrinosuppurative and hemorrhagic, diffuse, severe, with fibrin thrombi, epithelial and endothelial syncytia and eosinophilic intracytoplasmic inclusions, domestic short hair, feline.
Conference Note: Nipah virus is a single stranded RNA virus in the family Paramyxoviridae, subfamily Paramyxovirinae. Paramyxoviruses are negative sense RNA viruses that require RNA dependent RNA polymerase to be transcribed (transcription produces mRNA from DNA or RNA, translation produces protein from mRNA). Unlike orthomyxoviruses, paramyxoviruses have fusion protein (F protein). F protein is required for paramyxoviral penetration of host cells. Cellular proteases cleave F protein transforming it into F1 and F2 that act on the plasma membrane of the cell and cause fusion with the viral envelope. F protein is also important in maintaining infection by causing cell to cell fusion and allowing the virus to spread without exposure to neutralizing antibodies.
Hendra virus, another recently described paramyxovirus, was previously known as equine morbillivirus. In horses, Hendra virus can induce pulmonary edema characterized by gelatinous distention of the subpleural lymphatics. This gross appearance closely resembles African horse sickness. Histologically, there is vascular degeneration similar to equine viral arteritis, although endothelial syncytia are present in Hendra virus disease and absent in equine viral arteritis. Cats experimentally infected with Hendra virus can develop lesions similar to those reported in Nipah virus pneumonia.

Contributor: CSIRO Animal Health, Australian Animal Health Laboratory (AAHL), Private Bag 24 Geelong, Vic, Australia 3220
1. Anonymous: Exotic Animal Disease Bulletin, Nipah virus in Malaysia: latest news from the Australian Animal Health Laboratory. Aust Vet J 77:474-5, 1999
2. Hooper PT, Ketterer PJ, Hyatt AD, Russell GM: Lesions of experimental equine morbillivirus pneumonia in horses. Vet Pathol 34(4):312-322, 1997
3. Hooper PT, Westbury HA, Russel; GM: The lesions of experimental equine morbillivirus disease in cats and guinea pigs. Vet Pathol 34(4):323-329, 1997
4. Paton NI, Leo YS, Zaki SR, Auchus AP, Lee KE, Ling AE, Chew SK, Ang B, Rollin P, Umapathi T, Sng I, Lee CC, Lim E, Ksiazek TG: Outbreak of Nipah-virus infection among abattoir workers in Singapore. The Lancet 354:1253-1256
Case III - 99030112 (AFIP 2685771)
Signalment: 5-year-old, intact-male, English setter dog.
History: Animal presented with a 2-3 month history of diarrhea. Dog is cachetic but has a ravenous appetite. Animal has reportedly lost 20 pounds in past month.
Gross Pathology: No abnormalities noted following endoscopic examination.
Contributor's Diagnosis and Comments: Granulomatous enteritis with numerous intralesional and intracellular organisms consistent with Histoplasma capsulatum.
The intestinal villi are markedly widened and blunted by numerous inflammatory cells that have infiltrated the lamina propria. The macrophages contain numerous microorganisms characterized by a central, 1-2 micron basophilic spherule surrounded by a 1 micron, circumferential, clear capsule (consistent with Histoplasma capsulatum). Whereas the organisms predominate in the villi, they are also present within macrophages located deeper at the level of the crypts. Some sections exhibit hyperplasia of the crypt epithelial cells.
This case was exciting because of the tremendous numbers of the Histoplasma present in nearly every section. They are clearly visible without the need for special stains even at lower magnifications. Lastly, this case represents one of the rare rewards of making a significant diagnosis from an endoscopically collected biopsy.
AFIP Diagnosis: Small intestine: Enteritis, granulomatous, diffuse, moderate, with numerous intrahistiocytic yeast, English setter, canine, etiology consistent with Histoplasma capsulatum.
Conference Note: Histoplasma capsulatum is a dimorphic fungus. At 25°C it exists in the mycelium form and at 37°C in the yeast form. Other dimorphic fungi include Blastomyces dermatitdis, Sporothrix schenckii and Coccidioides immitis. Dogs and cats are the two domestic species that are most susceptible to infection, whereas birds do not become infected due to their higher body temperature, which prohibits fungal growth. The route of infection is by ingestion or inhalation of microconidia from contaminated soil. Once inhaled or ingested, the microconidia are phagocytized by macrophages where there transform to yeast that reproduce by budding. The yeast is then disseminated throughout the body within cells of the monocyte-macrophage system.
In dogs, histoplasmosis is often disseminated with frequent gastrointestinal tract involvement, as in this case. In cats, the disease is also often disseminated and produces a variety of nonspecific clinical signs. Frequent clinicopathologic abnormalities include nonregenerative anemia, neutrophilia, monocytosis, lymphopenia and eosinophilia. Rarely, yeasts are present within polymorphonuclear cells on peripheral blood smears.
Histologic diagnosis is made by finding the thin walled, 2-4mm diameter yeast in tissue section, often within macrophages. The yeast reproduce by single, narrow-based buds. The periodic acid-Schiff reaction and fungal stains can be used to help identify the organism. Other organisms that might be included in the differential diagnosis include Leishmania sp., Toxoplasma gondii, Cryptococcus neoformans, Blastomyces dermatitdis and Sporothrix schenckii.
Contributor: Oklahoma State University, College of Veterinary Medicine, 250 VetMed, Stillwater, OK 74078.
1. Greene CE: Infectious Diseases of the Dog and Cat, 2nd ed., pp. 378-372. WB Saunders Company, Philadelphia, PA, 1998
2. Jones TC, Hunt RD, King NW: Veterinary Pathology, 6th ed. pp. 519-522, Williams and Wilkins, Philadelphia, PA, 1996
3. Valli VEO, Parry BW: The Hematopoietic system. In: Pathology of Domestic Animals, vol. 3, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed., pp. 247-249, Academic Press, San Diego, CA, 1993
Case IV - 97-5113 (AFIP 2683740)
Signalment: Bovine feedlot steer, age unspecified, mixed breed beef.
History: One month history of respiratory disease, treated with multiple antibiotics including Micotil, Trivetrin and Nuflor. Recent onset of hyphema and hypopyon (bilateral) and uveitis. Chronic diarrhea and roughness in skin of interdigital cleft. The animal was euthanized.
Gross Pathology: Steer was in good nutritional condition. Both eyes contained blood and cloudy white material in the anterior chambers. Multifocal erosions were seen on the lateral surfaces of the tongue. Interdigital skin was rough, but no distinct erosions were found. The lungs were diffusely covered with fibrin, and moderately firm adhesions were found between the lungs, diaphragm, costal pleura and pericardial sac. Cranioventral portions of both lungs were consolidated and contained a large number of abscesses. Hilar lymph nodes were enlarged, red and edematous. There were multifocal 1-5mm long white steaks throughout the myocardium, and these were most numerous in the ventricles. A single 5mm firm focus was seen in the free wall of the left ventricle. All joints examined contained a small amount of fibrin.
Laboratory Results:
Clinical Pathology: CBC showed a neutrophilia with a left shift, increased fibrinogen interpreted to be due to chronic inflammation.
Bacteriology: Lung: 4+ Arcanobacterium pyogenes. Immunology: Heart: Bovine viral diarrhea virus POSITIVE, Lung: Mycoplasma bovis POSITIVE.
Contributor's Diagnoses and Comments:
Heart: Vascultis and perivasculitis, chronic, multifocal, severe.
Myocarditis, chronic, generalized, necrotizing, severe.
Myocardial parasitism, mild.
Lung (not submitted): Bronchopneumonia, chronic, locally extensive, suppurative, severe with bronchiectasis.
Vasculitis and perivasculitis, generalized, severe, chronic.
Lymphoid depletion, moderate to severe, generalized.
Vasculitis and perivasculitis were seen in nearly every organ examined in this steer including: eyes, kidney, intestine, heart, lung, and brain. Vessels were surrounded by or contained within their walls a moderate infiltration of lymphocytes and plasma cells. Frequently fibrinoid necrosis and hyaline degeneration were seen in affected vessels. Affected vessels were positive by immunohistochemistry for bovine viral diarrhea (BVD) virus. Positive staining for BVD virus was also detected in atrophied Peyers patches but was not present in the mucosa of the intestine, or elsewhere in the body. Only A. pyogenes was cultured from the lung of this animal, but lesions were positive for M. bovis by immunohistochemistry. Lesions seen in the tongue of this animal were typical of bovine papular stomatitis, rather than BVD.
Vasculitis is an unusual lesion reported in animals with mucosal disease, and has not been reported in the literature to be associated with acute BVD infection. A single mention is made in reference to this occurrence in association with clinical laminitits in an adult bovine but the circumstances are not well described. Animals with mucosal disease are reported to have profuse diarrhea, wasting, and frequently severe enteritis, which was not seen in this case. There have been some recent reports documenting high mortality rates in cattle with acute BVD infection, and this is speculated to be due to a strain with increased virulence. Antemortem serology and virus isolation which may have helped to differentiate these syndromes were not performed.
Lesions in the heart and elsewhere were so severe, that a differential diagnosis of malignant catarrhal fever was considered in this case. Other animals with similar presentations and lesions have been submitted for PCR for this herpesvirus and have consistently been negative. Immunohistochemistry in this animal was positive for BVD virus in affected vessels and in situ hybridization on a limited number of cases with similar lesions have demonstrated BVD virus exclusively in affected vessels.
Immunoperoxidase for BVD
Case 7-4. Heart. Note strong staining of arterial endothelial cells for BVD antigens.
The association of BVD with vasculitis and chronic pneumonia involving M. bovis has been noted by pathologists in this laboratory for several years. There are reports in the literature of BVD being associated with other viruses and bacteria in the "shipping fever complex" but no specific mention is made of M. bovis. The specific interaction is unknown at this time but is speculated to be immunosuppression and impaired leukocyte response. Pneumonia involving M. bovis is often associated with a number of other agents including Pasteurella sp. A. pyogenes, and occasionally H. somnus. Many of the animals infected with M. bovis also show a fibrinous arthritis and/or tenosynovitis (which was mild in this case).
AFIP Diagnosis:
1. Heart: Myocarditis, lymphoplasmacytic and histiocytic, necrotizing, multifocal, moderate, with vasculitis and mineralization.
2. Heart: Sarcocysts, few.
Conference Note: Bovine viral diarrhea virus is a member of the order Flaviviridae, genus pestivirus. Flaviviruses are 45-60nm, enveloped, icosohedral single stranded, positive-sense, RNA viruses.
Acute infection with BVD virus results in diarrhea in young animals; with intrauterine infection, there is a wide range of possible effects, depending on the age of the fetus and the nature of the virus. Intrauterine infection with noncytopathic (based on behavior in tissue culture) BVD virus before 80 days of gestation often results in death and resorption or abortion; between 80-125 days, results vary from fetal abnormalities (retinal dysplasia, cerebellar hypoplasia, hydrocephalus) to immunotolerance and persistent infection. Previously it was thought that a second infection with a cytopathic virus resulted in "mucosal disease", but it has recently been discovered that this second infection is actually a mutation from a noncytopathic to cytopathic biotype. Clinically, mucosal disease is characterized by diarrhea, nasal discharge, erosive and ulcerative stomatitis, dehydration and eventual death. The pestivirus genus also includes hog cholera virus and border disease virus in sheep. BVD virus infection in pregnant sows can cause fetal death and resorption
A differential diagnosis for bovine viral myocarditis discussed by conference participants included malignant catarrhal fever (MCF) and rinderpest. Lesions of MCF are characterized by marked perivascular and intramural infiltration of predominately large lymphocytes with large nuclei and prominent nucleoli. There is often an associated fibrinoid necrotizing vasculitis. The characteristic inflammatory infiltrates and vascular changes occur in almost all organs. Unlike rinderpest and BVD, the underlying lymphoproliferative nature of MCF often causes a prominent lymphocytic hyperplasia in multiple lymph nodes and prominent lymphoid follicles in the splenic white pulp. The vascular lesions are more consistently present and more severe in MCF than in BVD.
Rinderpest, a morbillivirus of the family Paramyxoviridae, causes necrosis of intestinal glands and Peyer's patches reminiscent of the gastrointestinal lesions of BVD. Rinderpest may also cause a necrotizing vasculitis; however, syncytial cells with eosinophilic intracytoplasmic inclusions are often seen histologically in cattle infected with rinderpest, and when present, distinguish it from BVD and MCF.
Contributor: Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, S7N 5B4 Canada.
1. Baszler TV, Evermann J, Kaylor PS, Byington TC, Dilbeck PM: Diagnosis of naturally occurring bovine viral diarrhea virus infection in ruminants using monoclonal antibody-based immunohistochemistry. Vet Pathol 32:609-628, 1995
2. Carmas S, van Dreumel T, Ridpath J, Hazlett M, Aves D, Dubovi E, Tremblay R, Bolin S, Godkin A, Anderson N, Severe acute bovine viral diarrhea in Ontario, 1993-1995. J Vet Diagn Invest 10:27-35, 1998
3. Jones TC, Hunt RD, King NW: Diseases caused by viruses. In: Veterinary Pathology, 6th ed. Williams and Wilkins. pp. 299-302, 1997
4. Jubb KVF, Kennedy PC, Palmer N: The alimentary system. In: Pathology of Domestic Animals, 4th ed., vol. 2, pp. 149-158, Academic Press Inc., 1993
5. Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ: Veterinary Virology, 3rd ed. Pp. 29, 322-323, 563-566. Academic Press, San Diego, CA, 1999
6. Richer L, Marois P, Lamontagne L: Association of bovine viral diarrhea virus with multiple viral infections in bovine respiratory disease outbreaks. Can Vet J 29:713-716, 1988.
7. Svensson C, and Bergsten C: Laminitis in young dairy calves fed a high starch diet and with a history of bovine viral diarrhea virus infection. Vet Rec 140:574-577, 1997
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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