AFIP Wednesday Slide Conference - No. 24
March 29, 2000

Conference Moderator:
Dr. Corrie Brown, Diplomate, ACVP
Chairman, Department of Pathology
College of Veterinary Medicine
University of Georgia
Athens, Georgia 30602-7388
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Case I - 99-6934C ( AFIP 2682159)
Signalment: Six-day-old, 1.0 kg, white, male, cross-breed pig.
History: This pig was a member of a group of four piglets submitted for necropsy from a farrowing operation where piglets aged 5 to 14 days old, stopped growing, and developed a mild to moderate diarrhea. Morbidity was high, but mortality was relatively low. The herd had recently had problems with colibacillosis in young pigs, but they had responded well to antibiotic therapy. This group of pigs was not responding to antibiotic therapy and many pigs were unthrifty or stunted.
Gross Pathology: The piglet was presented alive and in poor body condition with mild to moderate yellowish diarrhea. Intestinal con-tents were bright yellow and watery. The spiral colon had mild diffuse mesenteric edema. A moderate amount of curdled milk was present in the stomach. No other significant gross changes were observed in the carcass. Two other piglets from this submission had similar lesions and the remaining pig appeared normal.
Laboratory Results: CBC results indicated a high normal WBC count (21.6 X 103), but other parameters were normal. No viruses were detected on electron microscopic evaluation of feces. Fluorescent antibody examinations of intestines were negative for coronavirus and rotavirus; and lung was negative for PRRS. Intestinal culture revealed low numbers of K88+ E. coli. Fecal smears for coccidial oocysts were negative.
Contributor's Diagnoses and Comments:
1. Small intestine, enteritis, necrotizing with intralesional immature coccidia compatible with Isospora suis.
2. Small intestine, enteritis, with attaching bacilli.
Sections of small intestine (jejunum and ileum) have mild to moderate diffuse atrophy of villi with blunting and fusion of remaining villi. Villus lacteals are moderately dilated. Rare small crypt abscesses are present. There is erosion of the villus tips with occasional necrotic epithelial cells. Adjacent intestinal epithelial cells are often flattened or cuboidal. Moderate numbers of coccidial meronts, banana-shaped merozoites, developing oocysts and microgametocytes that are morphologically compatible with Isospora suis are present in intestinal epithelium. Inflammatory infiltrate in the intestine is minimal to mild and consists of occasional small aggregates of neutrophils. Rare sections of small intestine have occasional small clusters of small Gram negative bacilli attached to villus epithelium resembling E. coli. Sections of spiral colon had moderate diffuse submucosal edema with dilation of lymphatics and overgrowth of mixed bacilli within the lumen.
Isospora suis is known for its short prepatent period (5 days) and rapid sporulation (12 hrs). Isospora suis replicates in the epithelium of the distal third of villi primarily in the jejunum and ileum. The height of host cell infection is at 4 - 5 days. Diarrhea often begins at 5 to 10 days of age and many animals become unthrifty and/or stunted. Pigs usually become infected at 1- 2 days of age, possibly due to ingestion of feces from the dam or environmental contamination. The severity of infection is directly related to the size of the inoculum. In mildly affected individuals the immature coccidial forms of Isospora suis are often low in number and require fresh tissues and diligent evaluation to identify.
AFIP Diagnosis: Small intestine: Enteritis, subacute, diffuse, mild, with villar blunting and fusion, necrosis, and numerous intraepithelial coccidia, cross-breed, porcine.
Conference Note: Coccidians are obligatory intracellular protozoa that parasitize the intestinal mucosa of all animal species. The two genera of most concern in swine are Eimeria and Isospora. Other coccidian genera include Toxoplasma, Sarcocystis, Hammondia, Besnoitia, Cystoisospora, and Frankelia. The various species of Eimeria and Isospora are species-specific and are usually found in specific segments of intestine. The majority infect villous and crypt epithelial cells, although some species can be found in lacteal endothelial cells, the lamina propria and regional lymph nodes. Synergism with other pathogens may result in marked pathologic changes.
Isospora suis is a species specific coccidial pathogen of very young swine that replicates in the villar tips of the jejunum and ileum. Piglets have an age related susceptibility to infection and disease. Very young pigs (1-2 days) are highly susceptible to infection whereas 2-4 week old piglets are commonly resistant. A very short life cycle (as little as 5 days) for I. suis can lead to significant disease in piglets as young as 5 days.
Other pathogens causing diarrhea in very young pigs include E. coli (colibacillosis; a hypersecretory disease with minimal microscopic changes), rotavirus (most common cause of villar tip destruction), coronavirus (transmissible gastroenteritis; characterized by severe villous atrophy and epithelial cell necrosis), Clostridium perfringens (usually older pigs, causes full thickness hemorrhagic enteritis).
No enterocyte-adherent bacilli were observed in the sections examined at the conference.
Contributor: Veterinary Diagnostic and Investigational Laboratory, College of Veterinary, Medicine, University of Georgia, PO Box 1389, Tifton, Georgia 31793.
1. Barker IK, Van Dreumel AA, Palmer N: The Alimentary System. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed, vol. 2, pp. 304-306. Academic Press, San Diego, CA, 1993
2. Lindsay DS, Blagburn BL, Dubey JP: Coccidia and Other Protozoa. In: Disease of Swine, eds. Straw BE, D'Allaira S, Mengeling WL, Taylor DJ, 8th edition, pp.655-659. Iowa State University Press, Ames, Iowa, 1999
Case II - 19476 (AFIP 2684461)
Signalment: Adult, female, white-tailed deer (Odocoileus virginianus)
History: In 1997, a game farm in Iowa purchased 3 adult male white-tailed deer from Minnesota and placed them in a pen with 20 does. Over the course of 3 months, all of the bucks died without clinical signs noted prior to deaths. Necropsies were not performed on the first 2 due to autolysis. Samples of the third revealed pulmonary congestion and multifocal hepatic necrosis but no etiologic diagnosis. In April 1998, four months after the last buck died, a pregnant doe from the same pen died without history of illness and was submitted to the Iowa State University Veterinary Diagnostic Laboratory. Two more adult deer died, one in June and one in August, and were submitted for diagnosis. Both had pulmonary edema and similar microscopic findings. No further deaths have occurred. Elk in an adjacent but separate enclosure were not affected.
Gross Pathology: The doe was pregnant with two 4-5 kg fetuses. Body condition was excellent. Moderate hydrothorax and severe diffuse bilateral pulmonary edema were present. Marked distention of interlobular septa with fluid and abundant froth in trachea and bronchi were noted. No other gross lesions were detected.
Laboratory Results: Bacterial cultures of lung, liver, spleen, pleural fluid, and brain were negative. Immunohistochemistry of lungs revealed adenoviral antigens in scattered endothelial cells of lung, abomasal submucosa, and lymph node using polyclonal antisera against bovine adenovirus type 5. Transmission electron microscopy of lung from deparaffinized blocks revealed loosely arranged 68-69 nm diameter icosahedral structures with electron-dense cores consistent with adenoviral nucleocapsids.
Contributor's Diagnoses and Comments:
1. Lung: Endothelial degeneration, hypertrophy, and necrosis, acute, multifocal, with intranuclear inclusion bodies consistent with adenovirus (adenovirus hemorrhagic disease)
2. Lung: interlobular edema and fibrin, acute, severe.
The lesions in this case are consistent with the pulmonary lesions previously described in both natural and experimental cases in black-tailed deer. Adenovirus was isolated from the last case submitted using white-tailed deer lung cell cultures from a newborn. The virus was neutralized by antiserum to the California black-tailed deer adenovirus isolate and had a virtually identical restriction endonuclease pattern. Although recognized by antisera to bovine adenovirus-5, hexon gene sequence data indicate that the black-tailed deer adenovirus is closely related to bovine adenovirus-3.
The deer from the Iowa herd had all been purchased since 1995 from Minnesota, Michigan, Ohio, and Wisconsin. This is likely the first case of deer adenovirus hemorrhagic disease diagnosed in the eastern US, and the first in white-tailed deer. In contrast to the cases in black-tailed deer where fawns are primarily affected, the white-tailed deer deaths occurred in adults. No intestinal lesions were found in the white-tailed deer in contrast to the intestinal hemorrhage often noted in the black-tailed. Virus was identified in abdominal viscera of the white-tailed deer.
Pulmonary edema is also present in both bluetongue and epizootic hemorrhagic disease; these are the predominant differential diagnoses for adenovirus hemorrhagic disease.
AFIP Diagnosis: Lung: Vasculitis, multifocal, moderate, with endothelial intranuclear inclusion bodies, edema, and fibrin white tailed deer (Odocoileus virginianus), cervid.
Conference Note: Additional information on adenoviral epizootics in black-tailed and mule deer can be found in 1998-99 Wednesday Slide Conference, 13 Jan 99, Case II ( 98/98wsc16.htm.)
The causative agent of adenovirus hemorrhagic disease (AHD) belongs to the genus Mastadenovirus of the family Adenoviridae. Adenoviruses are non-encapsulated, icosahedral, double-stranded DNA viruses that range from 70-90 nm in diameter. Adenoviruses have a longer replication cycle than other viruses and are therefore more likely to develop large, dense intranuclear inclusions consisting of massive numbers of virions. Ultrastructurally, adenoviruses are arranged in a characteristic paracrystalline array. There are two antigenically distinct genera of adenoviruses, namely Mastadenovirus (mammals) and Aviadenovirus (birds). Adenoviruses are generally host specific, although some may infect animals closely related to the definitive host. For example, sheep can be infected with some serotypes of bovine adenovirus.

Generally, transmission is fecal-oral or direct contact via oro-nasal secretions. Adenoviruses are primarily epitheliotropic (respiratory and enteric) and less frequently endotheliotropic. Mammalian adenoviral infections are usually subclinical. Exceptions are infectious canine hepatitis (CAV-1) and infections in immunosuppressed and stressed animals. Marked cytomegaly with large basophilic inclusions are often noted in avian and ovine adenoviral diseases.

Although conference participants all favored AHD, the following differential diagnosis was discussed: epizootic hemorrhagic disease, bluetongue, heartwater and malignant catarrhal fever. Diagnosis depends on detection of adenovirus in endothelial intranuclear inclusions.
Contributor: Department of Veterinary Pathology, Iowa State University, College of Veterinary Medicine, Ames, Iowa 50011-1250
1. Lapointe J-M, Hedges JF, Woods LW: The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3. Arch Virol 144:393-396, 1999
2. Woods LW, Hanley RS, Chiu PH, Lehmkuhl HD, Nordhausen RW, Stillian MH, Swift PK: Lesions and the transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns. Vet Pathol 36:100-110, 1999
3. Woods LW, Hanley RS, Chiu PH, Lehmkuhl HD, Nordhausen RW, Stillian MH, Swift PK: Experimental adenovirus hemorrhagic disease in yearling black-tailed deer. J Wildl Dis 33:801-811, 1997
4. Woods LW, Swift PK, Barr BC, Horzinek MC, Nordhausen RW, Stillian MH, Patton JF, Oliver MN, Johnes KR, MacLachlan NJ: Systemic adenovirus infection associated with high mortality in mule deer (Odocoileus hemionus) in California. Vet Pathol 33:125-132, 1996
Case III - 01713 (AFIP 2677997)
Signalment: Two tooth (1.5 - 2 years), Awassi, male, sheep, Ovis aires.
History: Imran Awassi wearily stretched his neck above the dusty wall of his new prison encampment and cast a rheumy eye out over the other assembled flock. He was no longer blasé about his situation and days of travelling had taken their toll. All around him his new friends stood with heads hanging, some grinding their teeth. Others had mild diarrhoea. More than he could count had collapsed and been taken from the room by blue-overalled men who sweated profusely and appeared agitated. In the last few hours his toes had begun to throb and his paunch ached. He baa-leated to no-one in particular.
The above sheep was one of 250 Awassi, Merino and cross-bred sheep which died in a group of 30,000 sheep held in a quarantine feedlot in New Zealand prior to loading and shipment to Middle Eastern markets.
Gross Pathology: The outstanding gross lesion in the sheep was rumen distension with fermenting, acidic-smelling content in which undigested meal/concentrate was present. The carcass was dehydrated and all viscera congested. There was patchy reddening of rumen epithelium especially affecting the ventral surface. In these areas the papillae were uneven and erosions and ulcers, often with irregular and ill-defined margins, had formed. The submucosa was edematous (as in the present sample), and the reaction extended transmurally with haemorrhage, congestion, and serosal thickening and localised peritonitis with unorganised omental adhesions.
Laboratory Results: Ruminal pH 4.1, motile protozoa were not identifiable in rumen fluid.
Contributor's Diagnoses and Comments:
1. Rumen: Rumenitis, acute, diffuse, severe with intraepithelial and submucosal microabscessation and degeneration of superficial epithelium.
2. Rumen: Rumenitis, necrotizing with intralesional fungal hyphae.
3. Rumen: Vasculitis, necrotizing with thrombosis and intralesional fungal hyphae.
4. Rumen: Peritonitis, steatitis, granulomatous
Etiology: Ruminal acidosis with secondary mycosis consistent with zygomycosis.
Ruminal acidosis most commonly occurs as a consequence of ingestion of excessive quantities of fermentable carbohydrate, resulting in the explosive proliferation of lactic acid producing organisms. In the first instance this is mainly Streptococci sp. As the acidity of the rumen increases (pH less than 4.5) the normal ruminal microflora is destroyed and other lactobacilli proliferate and produce further excesses of lactic acid. This results in acidosis and hemoconcentration with hypovolemic shock, the later the result of osmotic imbalance in the rumen. Both factors contribute to the death of the animal.
In the present outbreak, the most severely affected sheep were from the Awassi groups, in fact those which had been preconditioned longest to concentrate feeding. This group ate the ration greedily, whereas others in less or non-affected groups refused it. The carbohydrate in this ration was assayed at 28.1 gm/l00 gm dry matter which is considerably in excess of the recommended level. Whether or not Awassi sheep are more susceptible to acidosis than the other breeds represented in the feedlot is unreported.
Secondary fungal infections, in this case probably a mucoraceous Zygomycetes, are common opportunistic invaders of the ruminal wall in this condition.
AFIP Diagnosis: Rumen: Rumenitis, necrosuppurative, transmural, diffuse, moderate, with necrotizing vasculitis, myriad bacteria, and numerous fungal hyphae, Awassi sheep, ovine.

Conference Note: The general gross appearance of "grain overload" is a distended rumen filled with fermenting, undigested grain which gives off an acidic smell and has a fluid-like consistency.
The contributor described the pathogenesis, but this was expanded on during conference: increased carbohydrates lead to increased dissociated volatile fatty acids (propionic, butyric and acetic) -> decreased pH 5.5-5.0 -> increased Streptococci -> decreased pH, due to lactic acid production; both D and L isomers, of which the D isomer is poorly metabolized and accumulates in systemic circulation -> death of streptococci and proliferation of lactobacilli<5.0 -> further decrease of pH due to larger amounts of lactic acid production -> ruminal atony and cessation of salivary secretion + increased ruminal organic acids (not lactic acid) -> osmotic fluid shift -> movement of fluid from blood into rumen -> hemoconcentration, severe dehydration, elevations in serum protein, inorganic phosphorous, lactate, pyruvate, and liver enzymes, anuria, circulatory collapse, and diarrhea) -> systemic acidosis + vesicular degeneration of surface stratified squamous epithelium accompanied by granulocytic infiltrates into the epithelium and vesicles -> invasion by fungi and Fusobacterium -> liver abscesses and possible systemic disease, to include polioencephalomalacia due to proliferation of thiaminase-producing bacteria.
Contributor: New Zealand Registry of Animal Pathology, Pathology Section, IVABS Massey University, Palmerston North, New Zealand
1. Barker IK, Van Dreumel AA: The Alimentary System. In: Pathology of Domestic Animals, eds. Jubb KVF, Kennedy PC, Palmer N, 4th ed. vol. 1, pp. 47-50. Academic Press, Inc, San Diego CA, 1993
2. Bruere AN, West DM. Metabolic diseases. In: The Sheep: health, disease and production, eds. Bruere AN, West DM, pp.191-192. Foundation for Veterinary Continuing Education of the New Zealand Veterinary Association, Massey University, Palmerston North, New Zealand, 1993
3. Slyter LL, Rumsey TS: The effect of coliform bacteria, feed deprivation, and pH on ruminal D-lactic acid production by steer on continuous-culture microbial populations changed from forage to concentrates. J Anim Sci 69:3055-3066, 1991
Case IV- H86-0130 (AFIP 2505039)
Signalment: 15-month-old female Bali cattle (Bos javanicus)
History: This Bali cow was experimentally infected with Jembrana disease virus (JDV) approximately 15 days prior to euthanasia and necropsy. Clinical signs of fever, malaise, anorexia, depression, and lymphadenopathy began developing at 5 days post inoculation.
Gross Pathology: Severe lymphadenopathy and splenomegaly, several ecchymotic hemorrhages on serosal surfaces particularly epicardium, pulmonary consolidation, and pale foci in most parenchymatous organs including liver, myocardium, kidneys and adrenals.
Laboratory Results: Hematological changes included severe lymphopenia and thrombocytopenia, and moderate neutropenia.
Contributor's Diagnoses and Comments:
1. Lung: pulmonary vascular lymphocytosis and monocytosis, atypical, multifocal, subacute, moderate, with mild arteriolar medial hypertrophy.
2. Lymph node: atypical lymphoid hyperplasia (not on all sections).
3. Kidney: interstitial vascular and perivascular lymphocytosis and monocytosis, atypical, multifocal, subacute, moderate, with glomerular hypercellularity, segmentation and occasional synechiae (not on all sections).
4. Liver: portal and sinusoidal lymphocytosis and monocytosis, atypical, multifocal, subacute, moderate (not on all sections).
Jembrana disease was first recognized in 1964 in Bali and is now endemic throughout parts of Indonesia. It is an acute, severe disease of Bali cattle (Bos javanicus) with a short incubation period of 5-12 days. Fever, lymphadenopathy and lymphopenia are the major clinical changes (Soesanto et al, 1990; Soeharsono et al., 1990). Pathological changes reflect an intense lymphoproliferative disorder (Dharma, et al., 1991). There is marked enlargement of lymph nodes and spleen, which feature proliferating lymphoblastoid cells in parafollicular areas, and follicular atrophy. A proliferative lymphoid and mononuclear infiltrate is also found in many other tissues, but the central nervous system is spared.
Many cases show unusual intravascular lesions in the lungs as observed in this case, which contain proliferative, immature blastic cells, that have been identified as macrophages by electron microscopy (Budiarso & Rikihisa, 1992). Severe cases often show pleomorphic, basophilic, intracytoplasmic inclusions, in the proliferative mononuclear cell population, although the nature of these inclusions is unclear and repeated attempts to demonstrate viral antigen within them have been unsuccessful. Very high titres of virus (up to 10 to the 8th ID50/ml) are found in the plasma during the febrile stage of the disease and a persistent viremia can be demonstrated for at least 25 months. Surviving animals show regression of lesions commencing about five weeks post-infection. Mortality is usually about 17% and relates to hemorrhages and multiple organ systems, although the precise cause of death remains obscure.
The etiological agent, Jembrana disease virus (JDV), has been recently identified as a lentivirus, closely related to bovine immunodeficiency virus (BIV), by nucleotide sequence analysis (Chadwick et al., 1995a,b). Lentiviruses are usually associated with chronic progressive diseases such as granulomatous encephalitis, pneumonia, arthritis, mastitis, etc. (CAEV, Visna virus) or AIDS (HIV, SIV and FIV). Except for the acute phase of EIAV infection, it is most unusual for a lentivirus to induce an acute disease with a short incubation period. It is precedent however, by the emergence of a strain of SIV from sooty mangabey monkeys (SIV-SMMPBj14), that is acutely pathogenic in macaques (Fultz, 1994), and produces lymphoproliferative changes that are remarkably similar to those observed with Jembrana disease.
AFIP Diagnoses:
1. Lung; kidney: Atypical lymphohistiocytic infiltrates, perivascular, intravascular and multifocal, with vasculitis, Bali cow (Bos javanicus), bovine.
2. Lymph node: Atypical lymphohistiocytic hyperplasia, diffuse.
3. Liver: Atypical lymphohistiocytic infiltrates, portal and sinusoidal, diffuse, moderate, with biliary hyperplasia and portal fibrosis.

Conference Note: Without the knowledge that the tissues were from a Bos javanicus, it would be very difficult to determine the etiology from this slide. Conference participants considered a wide variety of possible etiologies from malignant catarrhal fever (MCF) (Alcelaphine herpesvirus 1), to lymphoma, to theileriosis (East Coast fever). Most favored MCF. The primary histologic features that differentiate MCF from Jembrana disease (JD) are: JD consistently produces a distinct alveolar pneumonia, mesangial glomerulonephritis, and choroid plexus infiltration, but spares the remaining CNS; JD can produce granular intracytoplasmic inclusions; and JD does not cause the generalized mural vasculitis that is commonly reported in MCF.
Contributor: Murdoch University, School of Veterinary Studies, Murdoch, WA 6150, Australia.
1. Budiarso IT, Rikihisa Y: Vascular lesions in lungs of Bali cattle with Jembrana disease. Vet Pathol 29:210-215, 1992
2. Chadwick BJ, Coelen RJ, Sammels LM, Kertayadnya G, Wilcox GE: Genomic sequence analysis identifies Jembrana disease virus as a new bovine lentivirus. J Gen Virol 76:189-192, 1995
3. Chadwick BJ, Coelen RJ, Sammels LM, Kertayadnya G, Wilcox GE: Nucleotide sequence analysis of Jembrana disease virus: a bovine lentivirus
associated with an acute disease syndrome. J Gen Virol 76:189-192, 1995
4. Dharma DMN, Budiantono A, Campbell RSF, Ladds PW: Studies on experimental Jembrana disease in Bali cattle. J Comp Pathol 105:397-414, 1991
5. Fultz PN: SIV-SMMPBj14: an atypical lentivirus. Cur Top Microb Immuno 188:65-76, 1994
6. Murphy FA, Gibbs EBJ, Horzinek MC, Studdert MJ: Veterinary Virology, 3rd ed., p. 389. Academic Press, San Diego, CA, 1999
7. Soeharsono S, Hartaningsih N, Soetrisno M, Kertayadnya G, Wilcox GE: Studies of experimental Jembrana disease in Bali cattle. I. Transmission and persistence of the infectious agent in ruminants and pigs, and resistance of recovered cattle to reinfection. J Comp Pathol 103:49-59, 1990
8. Soesanto M, Soeharsono S, Budiantono N, Sulistyana K, Tenaya M, Wilcox GE: Studies on experimental Jembrana disease in Bali cattle.
Clinical signs and haematological changes. J Comp Pathol 103:61-71, 1990
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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