AFIP Wednesday Slide Conference - No. 18
26 January 2000

Conference Moderator:
LTC Crystal M. Briscoe
Diplomate, ACVP
US Army Medical Research Institute of Chemical Defense
Aberdeen Proving Ground, MD 21010-5425
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Case I - PAI/WSC#1 (AFIP 2686445)
Signalment: 24-month-old New Zealand white rabbit.
Gross Pathology: 9.5x7x8 cm tan mass replacing right kidney.

Contributor's Diagnosis and Comments: Nephroblastoma (syn: embryonal nephroma, Wilm's tumor)
Although reported as rare in all species except the pig and chicken, this tumor does occur with some regularity in rabbits. This case is a classic presentation--a large tumor with no metastatic lesions in a fairly young animal. Embryonal nephromas are second in incidence only to uterine tumors in rabbits according to Benirschke et al. in Pathology of Laboratory Animals.
AFIP Diagnosis: Kidney: Nephroblastoma, New Zealand white rabbit, lagomorph.

Conference Note: As mentioned by the contributor, nephroblastomas occur commonly in pigs and chickens. Nephroblastomas are true embryonal tumors that arise from primitive nephrogenic blastema. They can be unilateral or bilateral, and are frequently very large, often compressing adjacent tissue and filling the abdominal cavity. Histologically nephroblastomas are composed of three components: primitive glomeruli, abortive tubules, and loosely arranged spindle cell stroma (blastema) which can differentiate into neural tissue, fat, striated muscle, cartilage and bone. With the exception of the dog where up to 50% of nephroblastomas have been reported to metastasize, they are rarely malignant.
In humans, nephroblastoma is referred to as Wilm's tumor and is the most common primary renal tumor of childhood. Wilm's tumor has been associated with a defect in chromosome 11 band p13 at or near the Wilm's tumor locus. The risk for developing Wilm's tumor is increased with three groups of congenital malformations including WAGR syndrome, Denys-Drash syndrome, and Beckwith-Wiedemann syndrome.
Contributor: Pathology Associated International, 15 Wormans Mill Court, Frederick, MD 21701
1. Benirschke K, Garner FM, Jones TC: Pathology of Laboratory Animals, Vol II, pp. 1171-1172. Springer-Verlag, New York, NY, 1978
2. Kelly WR: The Liver and Biliary System. In: Pathology of Domestic Animals. eds. Jubb KVR, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 387-389. Academic Press, San Diego, , 1993
3. Schofield D, Cotran RS: Diseases of Childhood and Infancy. In: Robbin's Pathologic Basis of Disease, eds. Cotran RS, Kumar V, Collins T, 6th ed., pp. 487-489. WB Saunders Company, Philadelphia, PA, 1999
Case II - NADC BCS 99-1 (AFIP 2673504)
Signalment: Two 7-week-old, crossbred, female, porcine (domestic)
Caesarian derived, colostrum deprived (CDCD)
History: These pigs were experimentally inoculated with porcine circovirus (type II) and porcine parvovirus. They were euthanized and necropsied 32 days post inoculation. These lesions were incidental findings.
Gross Pathology: The aortic, right atrioventricular, and left atrioventricular valves were markedly enlarged with proliferative tissue on all leaflets. These masses were multiple, large, raised, friable, white to yellow, and firmly attached to the valve leaflets. The brain contained no grossly evident lesions. The kidney contained multifocal red foci throughout the cortex.
Laboratory Results: Streptococcus sp. was cultured from the heart valves and brain. White blood cell counts were <10,000 cells/ml (normal: 10-20,000/ml) on the day of necropsy.
Contributor's Diagnoses and Comments:
1) Brain: encephalitis, purulent, acute, moderate to severe, multifocal
2) Heart: endocarditis, valvular, proliferative, purulent, subacute, severe, multifocal to coalescing, with intralesional cocci and mineralization.
Etiology: Streptococcus sp.
This case represents a classic case of streptococcal infection in nursery age swine. Streptococcus suis is found in the normal flora of the tonsil and nasal cavity of swine. S. suis infections are characterized by one or a combination of the following: septicemia, bronchopneumonia, embolic pneumonia, endocarditis, polyarthritis, meningitis, encephalitis, nephritis, ophthalmitis, and polyserositis. We attribute the leukopenia in the face of this fulminating bacterial infection to the effects of the porcine circovirus infection. In our studies, we have found that the type II porcine circovirus causes leukopenia.
AFIP Diagnoses:
1. Heart, valve: Valvulitis, fibrinosuppurative, chronic, focally extensive, severe, with numerous cocci, domestic pig, porcine.
2. Brain: Meningoencephalitis, subacute, multifocal, moderate, with microabcesses.
3. Heart: Myocarditis, subacute, multifocal, mild.

Conference Note: Numerous Streptococcus species have been reported to infect domestic swine, but Streptococcus suis has classically been recognized as the most common cause of streptococcal meningitis and valvulitis. Two types of S. suis are recognized. Type one infects young piglets (2-3 weeks of age) and causes septicemia, synovitis and meningitis; type two is clinically and pathologically similar, but generally produces a larger range of lesions, as described in the contributor's comments.
S. suis is a Gram positive, a hemolytic, coccus that can be found singly, in pairs and in short chains. S. suis is spread by aerosol or contact. The organisms multiply in the tonsillar crypts. From there they enter the circulation and cause septicemia, synovitis, meningitis and other localized infections. S. suis has zoonotic potential.
Contributor: National Animal Disease Center, 2300 Dayton Avenue, P.O. Box 70, Ames, IA 50010
1. Sanford SE: Gross and histopathological findings in unusual lesions caused by Streptococcus suis in pigs: I. Cardiac lesions, II. Central nervous system lesions. Can J Vet Res 51:481-485, 1987
2. Sanford SE, Higgins R: Streptococcal Diseases. In: Diseases of Swine, 7th ed., pp. 588-590. Iowa State University Press, Ames, IA, 1992
3. Sanford SE, Tilker AME: S. suis associated diseases in swine: observations of a 1-year study. J Am Vet Med Assn 181:673-676, 1982
4. Taylor DJ: Pig Diseases, 6th ed., pp. 207-212. St. Edmundsbury Press, Bury St Edmunds, UK, 1995
Case III - W308/99 (AFIP 2694732)
Signalment: mixed ages and sexes of quail.
History: King and Brown quail were hatched and reared in a suburban backyard amongst a variety of other aviary birds. Signs of respiratory distress including ocular and nasal discharges, swelling of the tissues around the eyes and labored, open-mouthed breathing were first seen at 4 weeks of age. Affected birds lost weight and died. Treatment provided by the owner (doxycycline in drinking water plus chloromycetin eye ointment) provided temporary remission of clinical signs but with relapse proceeding to death on withdrawal of the medication. Of 115 King Quail hatched 61(53%) died or required euthanasia and of 49 Brown Quail hatched 48 (98%) died or required euthanasia.
Eight live quail were submitted for examination. One died soon after submission, the other birds had evidence of respiratory distress, high respiratory rate and marked respiratory effort, swelling around the orbit, photophobia, serous rhinitis and conjunctivitis. They were killed with halothane.
Gross Pathology: There were no significant gross lesions apart from swelling of the tissues around the eyes, serous discharges from the eyes and nose and a slight excess of serous fluid in the conjunctival sacs and nasal and infra-orbital sinuses. In some birds there were small scabs or thickenings in the skin of the face/beak suggesting dermatitis; otherwise the plumage was normal.
Laboratory Results:
There was a moderate growth of Pseudomonas aeruginosa and Proteus mirabilis from swabs of the infra-orbital sinus of each of 6 birds. Mycoplasma broths were overgrown with bacteria.
Contributor's Diagnoses and Comments:
1. Hyperplastic/dysplastic conjunctivitis, rhinitis and sinusitis associated with respiratory cryptosporidiosis.
2. Secondary bacterial (Pseudomonas aeruginosa and Proteus mirabilis) infection of sinuses, extension to other tissues with production of acute necrotizing osteomyelitis of facial/cranial bones and/or cellulitis in periorbital and associated tissues.
3. Mycotic stomatitis and dermatitis associated with Candida-like fungi.
The circulated slides are cross-sections of the nasal cavity/infra-orbital sinus and eyes of each of two of the affected quail. In both there is extensive cryptosporidial infection of the conjunctival, nasal and sinus epithelia with both hyperplasia and dysplasia of the affected epithelia, including change from the normal ciliated columnar mucus-secreting structure, loss of cilia and mucus secretory function, disorientation of cells from their normal orderly position and form and the formation of papillary proliferations. A variety of different life cycle stages of Cryptosporidium is present in/on the luminal surface of affected epithelial cells. There is mild to moderate heterophilic and lymphoplasmacytic leukocyte infiltration in the connective tissues underlying affected epithelia and the development and/or hypertrophy of lymphoid follicles in some places. Accumulations of mucoid cellular debris, exudate and organisms are present within sinuses.
Sections of one of the circulated cases also have evidence of acute necrotizing osteomyelitis within the medullary cavities/air spaces of facial and/or cranial bones. This is associated with the presence of large colonies of gram negative bacilli, possibly the P. aeruginosa and/or P. mirabilis cultured from the sinuses.
Sections of the other circulated case have evidence of a moderate to intense cellulitis including edema, fibrinous exudate and mixed leukocyte infiltrations extending through the connective tissues adjacent to affected conjunctivae and/or sinuses, possibly also (though not definitely demonstrated) the result of extension of the bacterial infection into these tissues. The nasal gland has evidence of mild inflammation with the accumulation of degenerate inflammatory debris within its ducts.
Sections also have evidence of proliferation of Candida-like fungal organisms in the superficial layers of oral mucosa and/or in the squamous layers of the beak, facial skin and external nares. Most affected birds had moderate to severe mycotic stomatitis, esophagitis and/or ingluvitis probably in consequence of the prolonged antibiotic treatments provided by the owner.
Most affected birds also had cryptosporidial infection of their trachea; some only mild infections, others with severe infections resulting in hyperplasia/dysplasia of the affected epithelium. The primary bronchi of some birds were also affected, but none had evidence of significant pneumonia or air sac disease.
Most of the quail also had cryptosporidial infections of the epithelial lining of their bursa of Fabricius, one with severe infection and hyperplasia/dysplasia of the affected epithelium, prominent heterophil infiltration in and through the epithelium and the accumulation of a cast of necrotic debris within the lumen of the bursa. While some quail had mild coccidial gametocyte parasitism of their intestinal mucosa none had evidence of cryptosporidial infection of intestinal epithelia.
Isolation of P. aeruginosa and P. mirabilis from the infra-orbital sinus of each of six birds implies a significant finding yet neither organism might have been expected as a primary pathogen in this site. Demonstration of respiratory cryptosporidiosis in each of the birds suggested it as the primary disease with the bacteria as secondary pathogens especially in those birds which developed cellulitis and/or osteomyelitis.
Differential diagnosis of the clinical problem required elimination of infections caused by Pasteurella sp., Haemophilus sp., Mycoplasma sp. and related bacteria. Mycoplasma broths were overgrown by bacteria thus precluding further exploration of their importance. None of the other bacteria considered to be possible significant pathogens were isolated in primary cultures. In the light of the histopathological findings it seemed reasonable to discount the possibility of their importance. Moreover microscopic examination of tissues failed to demonstrate evidence of any significant viral disease, particularly adenovirus which might have suggested a diagnosis of "quail bronchitis". No investigative service was available for further virological exploration of these birds. There was no evidence of mycotic respiratory disease in any of the quail.
Some of the affected quail had relatively normal lymphoid follicles in their bursa of Fabricius. In other birds the follicles were small and had a thin cortex but medullae were well populated with lymphocytes and there was only a minimum of interstitial fibrous tissue. In a few birds bursal follicles were very small to almost non-existent. It is unknown whether this implied any degree of immunocompromization; impressions were that the change represented an age-related atrophy of bursal follicles rather than the effects of an immuno-suppressive agent. Similarly the thymus of most birds, though small, was otherwise normal in structure and that this also was considered an age-related atrophy. The submitted specimens were from birds of different age and no "normal" tissues of these species of quail were available for comparison. The spleen of only some of the quail was available for microscopic examination; those examined appeared to have the normal range of lymphoid components and structure.
Both respiratory and intestinal cryptosporidiosis have been described in quail though definition of the specific organism(s) involved would seem to require further work (Current, 1997). Cryptosporidium baileyi is well recognized as the cause of cryptosporidial infections of the respiratory tract and bursa of Fabricius of domestic fowl and is associated with the development of lesions similar to those occurring in these quail. Similar respiratory and bursal cryptosporidiosis was reported in Coturnix coturnix quail in South Australia (Tham et al. 1982) but without characterization of the species involved. Intestinal cryptosporidiosis with clinical signs of diarrhea and lesions of enteritis was reported in bobwhite quail (Colinus virginianus) (Hoerr et al. 1985). There was no evidence of infection of the respiratory tract and/or bursa of Fabricius in those birds.
Further submissions from the affected group of these quail had similar clinical signs and lesions. Impression smears of their tracheal mucosa, stained by the acid-fast method, contained cryptosporidial oocysts suggestive of C. baileyi. DNA analysis, courtesy of Dr. Una Morgan, Murdoch University, Perth, Western Australia, was consistent with their identification as C. baileyi.
AFIP Diagnosis:
1. Conjunctiva, nasal mucosa, and sinuses: Conjunctivitis, sinusitis, and rhinitis, proliferative, subacute, multifocal, moderate, with numerous superficial epithelial adherent protozoa, king (Coturnix chinensis) and brown (Coturnix australis) quail, avian, etiology consistent with Cryptosporidium sp.
2. Periorbital tissue: Cellulitis, subacute, focally extensive, severe.
3. Turbinates: Osteomyelitis, heterophilic, multifocal, moderate, with bacilli.
4. Oral mucosa: Stomatitis, hyperkeratotic, multifocal, moderate, with superficial crust containing yeast, hyphae, pseudohyphae, and abundant cocci.
Conference Note: Cryptosporidium species are coccidian parasites that infect the microvillous borders of host epithelial cells. In birds they infect the respiratory, intestinal and bursal epithelium. Species of Cryptosporidium have also been reported in a wide variety of vertebrate hosts, including reptiles, amphibians, fish, and mammals (including humans). C. parvum infects only mammals, C. baileyi and C. meleagridis infect only birds, and C. serpentis infects only reptiles.
Cryptosporidium spp. are apicomplexan protozoans in the Order Eucoccidiorida, Family Cryptosporidiidae. Transmission is direct. Sporulated oocysts are excreted in the feces. Ingested or inhaled sporozoites invade epithelial cells, disrupting the apical microvillous border in an intracellular but extracytoplasmic location. Asexual replication results in formation of first, second and third generation meronts (merogony). Merozoites then form microgamonts and macrogamonts (gametogony), which fuse (fertilization) forming an oocyst. Formation of sporulated oocysts (sporogony) occurs within the host epithelial cell.
Contributor: School of Veterinary Science, University of Melbourne, Veterinary Clinical Centre, Werribee Victoria 3030, Australia
1. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues. 2nd ed., pp. 37-40. Armed Forces Institute of Pathology, Washington, DC, 1998
2. Current WL: Cryptosporidiosis. In: Diseases of Poultry, ed. Calnek BW, 10th ed., pp. 883-890. Mosby-Wolfe, London, 1997
3. Hoerr FJ, Current WL, Haynes TB: Fatal Cryptosporidiosis in Quail. Avian Dis 30:421-425, 1986
4. Tham VL, Kniesberg S, Dixon BR: Cryptosporidiosis in Quails. Avian Pathol 11:619-626, 1982
Case IV - 96-2874 (AFIP2592869)
Signalment: Adult male mixed breed dog.
History: This dog was one of five received by University Animal Care for use in an experimental protocol. Routine physical exam and screening exams were performed. Babesia canis and Babesia gibsonii parasites were seen on blood smears. The dogs were administered di-(p-amidinophenyl) triazene (Berenil®) at a dosage of either 7mg/kg IM one time or 3.5mg/kg IM SID on two consecutive days to clear the infection. Twenty-four to 48 hours post injection, the dogs developed clinical signs, which included head tilt, ataxia, vomiting, fever, diarrhea, intention tremors, opisthotonous and seizures. A neurologic examination of the dog from which slides for this conference were obtained revealed exaggerated extensor radialis and patellar reflexes on the right side, normal pupillary reflexes, absent proprioceptive reflexes in all four limbs, and exaggerated hopping reflex to the side in all four limbs. The dogs were euthanized and necropsies performed.
Gross Pathology: Foci of hemorrhage and malacia were observed in the cerebellum and medulla in the location of the cerebellar and vestibular nuclei.
Laboratory Results: A complete blood count, serum chemistry panel and urinalysis were unremarkable. Liquid chromatography-mass spectrometry revealed a three-fold, higher level of di-(p-amidinophenyl) triazene in the lot of Berenil® administered to these animals when compared to an older lot which had been used previously without incident. No evidence of product degradation was apparent in either lot. Quantitation of the drug concentration was not possible due to our inability to obtain a standard.

Contributor's Diagnosis and Comments: Necrosis, hemorrhagic, subacute, multifocal, severe, vestibular and cerebellar roof nuclei, brain.

Etiology: di-(p-amidinophenyl) triazene toxicity.
Di-(p-amidinophenyl) triazene is one of a group of drugs known as diamidines. These were developed specifically for their antitrypanosomal activity. Some were also found to be effective against Babesia sp. and, outside of the United States, are widely used for treatment of this infection in dogs. The drug is not available in the United States but can (and was in this case) be purchased from pharmacies in Mexico.
Reports of toxicity following therapeutic use of di-(p-amidinophenyl) triazene have occurred sporadically in the literature and one experimental replication study has been published. Typically, the history is that of repeated therapeutic doses followed in a few days by the acute onset of CNS signs. At the recommended dosage rate of 3.5 mg/kg for elimination of Babesia canis, serious side effects are not expected. However, there have been undocumented reports of dogs treated at this dosage that have developed clinical signs, died and had characteristic necropsy lesions. The dosage of 7mg/kg was utilized for treatment of our dogs based on recommendations in a current pharmacology text for the elimination of Babesia gibsonii. It is recommended that the dosage never exceed 7mg/kg, single dose, administered IM. Dosage over 10mg/kg will result in CNS injury. There is, apparently, marked individual variability in sensitivity to the drug among dogs. The results of the LC-Mass Spec analysis suggest that the lot used to treat our dogs contained a higher concentration of the drug than represented on the label.
Clinical signs of toxicity include behavioral changes, nystagmus, ataxia, extensor rigidity, opisthotonus, coma and death. Hemorrhagic gastroenteritis, hepatic failure and muscle degeneration have also been reported. Necropsy lesions are foci of hemorrhage and malacia affecting white and gray matter in the cerebellum, medulla oblongata, midbrain and thalamus. The vestibular nuclei and cerebellar roof nuclei are the most commonly involved areas. Less frequently affected areas include the olfactory lobes, basal ganglia, thalamus, midbrain and cerebellar peduncles and cortex (uvula). The histologic lesions are multifocal hemorrhage, necrosis, edema and rarefaction followed by an influx of neutrophils and Wallerian degeneration. The mechanism of toxicity is not well defined. The drug has a histamine-like action which leads to increased vascular permeability and may have some relevance to the lesions in the CNS.
AFIP Diagnosis: Cerebellum and brainstem, cerebellar nuclei: Necrosis and hemorrhage, bilaterally symmetrical, extensive, with neutrophilic inflammation, fibrinoid vasculitis, and perivascular edema, mixed breed, canine.

Conference Note: The differential diagnosis for necrosis and hemorrhage of cerebellar nuclei includes thiamin defiency, nigropallidal encephalomalacia in horses, focal symmetrical spinal poliomalacia of sheep and goats, nicotinamide antagonists, polioencephalomalacia of ruminants, Aspergillus clavatus mycotoxicosis, and rickettsial infection. In very few of these conditions is the pathogenesis well understood. A common pathway seems to be shared by many of these diseases, whereby ischemia, hypoxia or a direct toxic effect results in lactic acid production and/or tissue damage with release of vasoactive amines, leading to vasodilatation, edema, and inflammation.
CNS edema results from 4 mechanisms:
1. increased vascular permeability, resulting in increased extracellular fluid (vasogenic type);
2. increased intracellular fluid with intact vascular permeability (cytotoxic type);
3. increased hydrostatic pressure (hydrostatic type);
4. abnormal osmotic gradient between tissues and blood (osmotic type).
CNS cells and tissue structures, in decreasing order of susceptibility to ischemia are neurons, oligodendroglia, astrocytes, microglia, and blood vessels. Neuronal susceptibility to ischemia varies by location within the CNS.
Contributor: Arizona Veterinary Diagnostic Laboratory, University of Arizona, Tucson, Arizona 85705

1. Jubb KVF, Huxtable CR: The Nervous System. In: Pathology of Domestic Animals, eds. KVF Jubb, PC Kennedy, N Palmer, 4th ed., vol. 1, pp. 338-350. Academic Press, San Diego, CA, 1993
2. Carlton WJ, McGavin MD: Thompson's Special Veterinary Pathology, 2nd ed., pp. 338-350. Mosby Yearbook, Inc., St Louis, MO, 1995
3. Moore DJ: Therapeutic implications of Babesia canis infection in dogs. J South African Vet Assoc 50:346-352, 1979
4. Naude TW, Basson PA, Pienaar JG: Experimental diamidine poisoning due to commonly used babecides. Onderstepoort J Vet Res 37:173-184,1979
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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