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CASE I – UFSM-1 (AFIP 2741090) plus gross photo
Signalment: 6-year-old, female, quarter horse, equine
History: This horse had a unilateral mass in the nasal cavity. The mass was surgically excised, fixed in 10% formalin, and sent to our laboratory.
Gross Pathology: The submitted specimen was a firm, pink, 3 x 2 x 3 cm mass. The external surface was lobulated, irregular and cauliflower-like with a smooth and gray cut surface. White pinpoint foci could be seen in the cut surface.
Laboratory Results: None
Contributor’s Diagnosis and Comment: Nasal cavity, focal granulomatous rhinitis with pseudoeptheliomatous hyperplasia and squamous metaplasia of the surface epithelium associated with organisms with morphology consistent with Rhinosporidium seeberi.
Microscopically, the mass consists of fibrovascular tissue infiltrated with macrophages, lymphocytes and neutrophils and covered by a pseudo-epitheliomatous and metaplastic epithelium. Within this mass, numerous spherical organisms with a thick and double contoured wall can be seen. The larger ones (sporangia) are 300 m m in diameter and contain endospores that are 2-7 m m in diameter. These elements represent the different evolutive stages of development of Rhinosporidium seeberi.
Rhinosporidiosis is a chronic infection of mucous membranes, mainly of the nasal cavity, caused by R. seeberi. The disease was first reported in humans at the end of the nineteenth century. Carlos Seeber described the etiologic agent in 1900 and it was classified as a fungus in 1923. The first report of rhinosporidiosis in animals dates from 1913. The disease has been sporadically reported in many countries in several animal species, including dogs, horses, donkeys, cattle, cats, geese, ducks, and man. Endemic areas exist in Sri Lanka, India and Argentina. In Brazil, there are reports of rhinosporidiosis in man, as well as 16 published reports in animals, to include horses, mules and cattle. The source of infection of rhinosporidiosis is not clearly determined, but it seems associated with stagnant water. Trauma can be a predisposing factor to the infection. In cattle, an association of rhinosporidiosis to nose puncture for placement of nose rings and to the infection by Schistosoma nasalis has been reported.
In animals, the disease has been described mainly as polyps in the nasal cavity. Clinically, affected animals show mucopurulent nasal discharge that is occasionally tinged with blood. If the polyps are large enough to obstruct the nasal cavity, dyspnea and stertorous breathing may be present. The general health of the animal is usually not affected by the growths in the nasal cavity, and the dissemination of the infection to internal organs has not been reported. There are reports of polyps developing in the conjunctival sac, vagina, and several sites in the skin (particularly the ears). Polyps usually, but not always, are single and unilateral and may be sessile or pedunculated. Their shape is irregular (occasionally cauliflower-like) and their size is variable (usually 2-3 cm). The masses are soft, pink, and bleed easily. The multiple white pinpoints that can be seen in the external and cut surfaces correspond to mature sporangia.
Histologically, the polyps consist of fibrovascular or myxoid stroma within which R. seeberi organisms appear as spherical bodies of varying sizes. This stroma is covered by a usually intact epithelium, which occasionally is hyperplastic and contains sporangia. A sporangium (the mature organism) measures up to 300 m m in diameter and contains many endospores (2-10 m m in diameter). Endospores are larger and more uniformly round towards the center of the sporangium. Smaller, usually flattened, endospores are localized at the periphery. Upon rupture of the sporangium cell wall, endospores are released and develop to trophocytes. Trophocytes are up to 100 m m in diameter, contain a single nucleus with a prominent nucleolus, and are thought to mature to the sporangia containing endospores. Although special methods such as PAS, Gomori’s methenamine silver and Mayer’s mucicarmine can be used to stain the different stages of maturation of this organism, R. seeberi is readily identified in sections stained by H&E.
Histologically, the tissue reaction and the morphology of the agent in rhinosporidiosis may resemble those of coccidiodomycosis but can be distinguished by the following aspects: i) mature sporangia of R. seeberi are larger and contain endospores in varying stages of development which are larger than those of Coccidioides immitis; ii) PAS stains only the walls of C. immitis, whereas it stains the whole endospore of R. seeberi; and iii) only the endospores of R. seeberi contain inner granules.
Clinically, nasal rhinosporidiosis in horses should be differentiated from congenital nasal cysts, other inflammatory polyps, neoplasms, progressive ethmoid hematoma, and other granulomatous lesions caused by fungi such as Coccidioides immitis, Cryptococcus neoformans, Histoplasma spp., Aspergillus spp., Pseudoallescheria boydii, Conidiobolus coronatus, and Pythium insidiosum.
Treatment consists of surgical removal and cauterization of the base of the lesion.
AFIP Diagnosis: Nasal mucosa: Inflammatory polyp, with sporangia and trophocytes, quarter horse, equine, etiology consistent with Rhinosporidium seeberi.
Conference Comment: Rhinosporidium seeberi formerly was classified as a fungus. Recent analysis of the organism using polymerase chain reaction has shown it to be a protist in the DRIPs clade. DRIPs is a novel group of aquatic protistan parasites of fish and amphibians that are near the animal-fungal phylogenetic divergence. DRIPs takes its name from the organisms Dermocystidium spp., the Rosette agent, Ichthyophonus spp. and Psorospermium spp. The name Ichthyosporea has been proposed for this expanding taxon of microbes. Dermocystidium salmonis, the closest known relative to R. seeberi, causes gill inflammation and epithelial hyperplasia with large spherical structures containing endospores in salmonids. Prototheca spp., Chlorella, and Coccidioides immitis also reproduce by endosporulation.
Rhinosporidium seeberi infection has been reported in the United States as a sporadic emerging human and animal pathogen. The link between disease and exposure to water has led researchers to hypothesize that the natural hosts of R. seeberi are fish or other aquatic animals. The organism has not been cultured in cell-free media, but has been cultured in monolayers of human rectal tumor cells.
Contributor: Universidade Federal de Santa Maria, Departamento de Patologia, 97105-900, Santa Maria, RS, Brazil
References: 1. Chandler FW, Kaplan W, Ajello L: Rhinosporidiosis. In: A colour atlas and textbook of the histopathology of mycotic diseases, pp. 109-111, Wolf Medical Publications, Weert, 1980
2. Dungworth DL: The respiratory system. In: Pathology of domestic animals, ed. Jubb KVF, Kennedy PC, Palmer N, 4th ed., vol. 2, pp. 561, Academic Press, San Diego, CA, 1993
3. Easley JR, Meuten DJ, Levy MG, Dykstra MJ, Breitschwerdt EB, Holzinger EA, Cattley RC: Nasal Rhinosporidiosis in the Dog. Vet Pathol 23:50-56, 1986
4. Fredricks DN, Jolley JA, Lepp PW, Kosek JC, Relman DA: Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites. Emerg Inf Dis 6(3):273-282, 2000
5. Jones TC, Hunt RD, King NW: Diseases caused by fungi. In: Veterinary Pathology, 6th ed., pp. 526-527, Williams & Wilkins, Baltimore, MD, 1997
6. Jungerman PF, Schwartzman RM: Rhinosporidiosis. In: Veterinary medical mycology, pp.40-47, Lea & Febiger, Philadelphia, PA, 1972
7. Londero AT, Santos MN, Freitas JB: Animal Rhinosporidiosis in Brazil. Report of three additional cases. Mycopathologia 60:171-173,1977
8. Robertson JT, Fenger CK: Diseases of the Nasal Passage. In: Current therapy in equine medicine 3, ed. Robison NE, pp. 268, WB Saunders Company, Philadelphia, PA, 1992
9. Timoney JF, Gillespie JH, Scott FW, Barlough JE. Hagan’s and Bruner’s microbiology and infectious diseases of domestic animals with reference to etiology, epizootiology, pathogenesis, immunity, diagnosis, and antimicrobial susceptibility, p. 415, Comstock Publishing Associates, Ithaca, NY, 1988
CASE II –00R453A (AFIP 2739545)
Signalment: Adult, female, domestic rabbit (Oryctolagus cuniculi)
History: Tissues are from a domestic rabbit that was housed in a hutch in a rabbitry in rural Iowa and died without clinical signs. A free roaming rabbit was found dead. One week later, rabbits that were housed in hutches started dying. Most were found dead without prior signs. This animal was necropsied by a private veterinarian. Tissue samples were sent to the state veterinary diagnostic laboratory. Ultimately, 25 of 27 rabbits from the rabbitry died.
Gross Pathology: The local practitioner reported finding pulmonary petechiae and food in the stomach. Liver lesions were not noted.
Laboratory Results: A mycotoxin screen on feed samples was negative. Bacterial cultures were also negative. A tentative diagnosis of rabbit calicivirus disease was made based on the microscopic hepatic lesions, and tissues were sent to USDA’s Foreign Animal Disease Diagnostic Laboratory for confirmation.
Rabbit calicivirus disease was diagnosed based on positive hemagglutination tests, viral particles consistent with calicivirus by electron microscopy, and characteristic histologic lesions. Two of three rabbits challenged with tissue homogenates from the dead rabbit and developed subsequent characteristic lesions. With immunohistochemistry, liver sections from these rabbits were weakly positive for rabbit calicivirus. PCR done on the tissue by the Instituto Nacional de Investigaciones Agrarias in Spain was also positive.
Contributor’s Diagnoses and Comment: 1. Liver: necrosis, coagulation, diffuse to submassive, acute, severe with apoptosis.
2. Lung: hemorrhage, multifocal, moderate, acute with occasional thrombi.
Very few hepatocytes remain viable in the section. The severe loss of hepatocytes that occurs as a result of calicivirus infection is thought to lead to disseminated intravascular coagulation (DIC) and hemorrhage. The cause of the DIC may be release of large amounts of thromboplastins due to the hepatolysis. Consumption of coagulation factors and acute hepatic deficiency could exacerbate the DIC. Hepatocellular death occurs via the apoptotic pathway and is unaccompanied by significant inflammation. Apoptotic changes have also been reported in macrophages, endothelial cells, circulating monocytes, lymphoid organs, and heart.
This is the first reported case of rabbit hemorrhagic disease (RHD), also termed rabbit calicivirus disease (RCD), in North America. After RHD was confirmed, the two surviving rabbits at this location were euthanatized, hutches were burned and buried, and premises were disinfected. Four months later, the source of the virus remained undetermined. No new live rabbits had been introduced onto the premises in the previous 2 years; and no live rabbits had left the premises and returned for the previous 7 months. No subsequent cases have been reported.
RHD, which affects only rabbits of the Oryctolagus genus and not cottontails (Sylvilagus sp.) or jackrabbits, started in China in the early 1980s, likely associated with rabbits imported from Europe. The disease killed thousands of rabbits in Asia and spread to Europe where it killed many native wild rabbits. The virus was reported in Mexico in 1988 and was apparently eradicated by 1992. The virus was studied extensively in Australia for its affects on other species, including humans, and was shown not to infect any other tested mammals on that continent.
In Australia, rabbit overpopulation has led to severe environmental degradation, particularly of native fauna. Field trials assessing the use of calicivirus as a potential biologic population control agent were conducted on Wardang Island off the coast of Australia. In 1995, the virus escaped from Wardang Island and spread to the mainland where it progressed rapidly throughout the country killing millions of rabbits. The use of calicivirus as a biologic control agent has produced mixed results and remains very controversial.
There is some variation among RHD viruses. The isolate from Iowa was not as closely related to the Australian virus (which came from a Czech isolate) as it is to other European strains. A non-pathogenic rabbit hemorrhagic disease-like virus has been reported in Italy.
AFIP Diagnoses: 1. Liver: Hepatocellular dissociation, diffuse, with necrosis/apoptosis and hemorrhage, rabbit (Oryctolagus cuniculi), lagomorph.
2. Lung: Edema, alveolar, diffuse, with multifocal hemorrhage and mild, acute interstitial pneumonia.
Conference Comment: The pathologic changes present in rabbit hemorrhagic disease, characterized by acute liver damage and disseminated intravascular coagulation, have been reported to be due to apoptosis rather than necrosis. Programmed cell death is a common cellular response to control RNA viral replication and growth. Virus specific genes have been identified that can prolong host cell survival to assure completion of the viral life cycle.
Ultrastructurally, apoptotic cells undergo shrinkage, chromatin margination, cytoplasmic membrane blebbing, and nuclear condensation and segmentation with division into apoptotic bodies that can then be phagocytized. Detection and confirmation of apoptosis by terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) technique demonstrates internucleosomal double strand breaks of DNA. The TUNEL assay, combined with electron microscopy, can definitively distinguish apoptosis from necrosis.
Disseminated intravascular coagulation is characteristic of RHD. Apoptosis of intravascular monocytes has been suggested as a possible initiator of this process.
Contributor: Iowa State University, Department of Veterinary Pathology and the ISU Veterinary Diagnostic Laboratory, Ames, IA
References: 1. Alonso C, Oviedo JM, Martin-Alonso JM, Diaz E, Boga JA, Parra F: Programmed cell death in the pathogenesis of rabbit hemorrhagic disease. Arch Virol 143:321-332, 1998
2. Capucci L, Nardin A, Lavazza A: Seroconversion in an industrial unit of rabbits infected with a non-pathogenic rabbit haemorrhagic disease-like virus. Vet Rec 140:647-650, 1997
3. Guittre C, Ruvoen-Clouet N, Barraud L, Cherel Y, Baginski I, Prave M, Ganiere JP, Trepo C, Cova L: Early stages of rabbit haemorrhagic disease virus infection monitored by polymerase chain reaction. J Vet Med 43:109-118, 1996
4. Park JH, Lee Y-S, Itakura C: Pathogenesis of acute necrotic hepatitis in rabbit hemorrhagic disease. Lab An Sci 45(4):445-449, 1995
5. Plassiart G, Guelfi J-F, Ganiere J-P, Wang B, Andre-Fontaine G, Wyers M: Hematological parameters and visceral lesions relationships in rabbit viral hemorrhagic disease. J Vet Med 39:443-453, 1992
6. Ramiro-Ibanez F, Martin-Alonso JM, Palencia PG, Parra F, Alonso C: Macrophage tropism of rabbit hemorrhagic disease virus is associated with vascular pathology. Virus Res 60:21-28, 1999
CASE III – LOT006 (AFIP 2741626)
Signalment: Rats, age and sex unknown
History: The attending veterinarians detected clinical signs of chromodacryorrhea, blepharospasm and head swelling in multiple rats.
Gross Pathology: None reported.
Laboratory Results: None provided.
Contributor’s Diagnosis and Comment: Harderian Gland: Marked, diffuse, subacute, necrotizing and suppurative adenitis with multifocal regeneration and squamous metaplasia.
Etiology: Sialodacryoadenitis virus (SDAV) infection
The architecture of the Harderian gland is diffusely and severely disrupted by variably sized and coalescing areas of coagulative necrosis of ducts and acini. There are accumulations of cellular debris, porphyrin pigment and infiltrates of mostly macrophages, neutrophils, lymphocytes and plasma cells (the lymphoid cells being more prominent at the periphery of the necrotic gland). There is mild to moderate, diffuse, interstitial edema with separation of the necrotic glandular lobules by a protein-rich edema fluid, vascular ectasia and scattered small areas of hemorrhage. Blood and lymphatic vessels are diffusely reactive and lined by prominent and hyperplastic endothelial cells. Multifocally, there is evidence of ductal and acinar regeneration characterized by tightly packed, large basophilic epithelial cells with prominent euchromatic nuclei and evidence of ductal and acinar squamous metaplasia (variable severity among sections).
The attending veterinarians detected clinical signs consistent with SDAV infection in multiple rat rooms in one of our facilities. The rats shipped to our facility were most likely exposed through secondary transmission to SDAV. Harderian glands (histology) and serum (serology) were collected from selected rats to confirm SDAV infection. After SDAV infection was confirmed, the affected facility was depopulated.
SDAV is a rat coronavirus that causes transient necrotizing and inflammatory lesions of salivary and lacrimal glands and upper respiratory tract (1). In addition, reproductive disorders, behavioral changes, and depletion of epidermal growth factor (EGF) from submandibular salivary glands have been associated with SDAV infection.
The disease is very contagious and SDAV is mostly transmitted from rat to rat through fomites or handling. Albeit unlikely with modern housing and husbandry techniques, rat to mouse transmission has been shown to occur.
The disease may be a subclinical infection or may lead to clinical signs indicative of salivary gland, lacrimal gland, and upper respiratory tract infection (such as sniffling, sneezing, blepharospasm, chromodacryorrhea, cervical edema, photophobia, etc.). Serologic surveys indicate that coronaviral infections are not infrequent in laboratory rats housed in research facilities. The disease is associated with high morbidity but low mortality. In euthymic rats, the disease is acute and self-limiting with shedding of virus occurring for approximately 1 week after infection. Athymic rats, however, appear to become chronically infected with persistence of the virus in salivary glands and respiratory tract for several months following experimental infection, indicating that normal T cell function is required for host defenses against SDAV.
In euthymic rats, acute lesions consist mostly of coagulative necrosis and inflammation of affected tissues. After a week of infection, reparative processes are evident. In the Harderian gland, one of these regenerative processes, squamous metaplasia of ductal and acinar structures, is considered almost pathognomonic of SDAV infection. After 3 to 4 weeks, regeneration in the affected tissues is almost complete with only focal residual lesions. In athymic rats, active lesions are seen for up to 6 months and bronchopneumonia can be observed. A diagnosis of SDAV infection is based on serology, histology and immunohistochemistry.
AFIP Diagnosis: Harderian gland: Dacryoadenitis, necrotizing, subacute, diffuse, severe, with squamous metaplasia, rat, strain not specified, rodent.
Conference Comment: In addition to the lesions described by the contributor, ocular changes such as keratitis sicca, corneal ulceration, uveitis and megaloglobus can be seen secondary to lacrimal gland dysfunction. Neonatal rats may develop pneumonia and olfactory dysfunction that can lead to nursing failure and death.
Contributor: Pharmacia/Searle, 4901 Searle Parkway, Skokie, IL 60077
References: 1. Bihun CGD, Percy DH: Morphologic changes in the nasal cavity associated with sialodacryoadenitis virus infection in the Wistar rat. Vet Pathol 32(1):1-10, 1995
2. Hajjar AM, DiGiacomo RF, Carpenter JK, Bingel SA, Moazed TC: Chronic sialodacryoadenitis virus (SDAV) infection in athymic rats. Lab Anim Sci 41(1):22-25, 1991
3. La Regina M, Woods L, Klender P, Gaertner DJ, Paturzo FX: Transmission of sialodacryoadenitis virus (SDAV) from infected rats to rats and mice through handling, close contact, and soiled bedding. Lab Anim Sci 42(4):344-346, 1992
4. Weir EC, Jacoby RO, Paturzo FX, Johnson EA, Ardito RB: Persistence of sialodacryoadenitis virus in athymic rats. Lab Anim Sci, 40(2):138-143, 1990
CASE IV – 99198 (AFIP 2738707)
Signalment: Fourteen-year-old, male, African green monkey (Cercopithecus aethiops)
History: This animal was one of several affected animals out of a larger group that were housed in adjoining rooms. Clinical signs included lethargy, hyperpnea and loss of appetite. This animal’s health declined despite antibiotic treatment and supportive care, and euthanasia was elected.
Gross Pathology: The lungs were diffusely mottled dark red to brown, and were heavy and wet; foam exuded from major airways and cut surfaces of the lung. The lungs did not float in formalin. There was scant, red, watery fluid in the pleural space (10 ml).
Laboratory Results: Culture of Bordetella bronchiseptica from lung, pericardium, and pleural space.
Contributor’s Diagnosis and Comment: Lung: Severe, diffuse, acute, necrotizing, suppurative bronchopneumonia, with intralesional bacterial colonies.
Etiology: Bordetella bronchiseptica
Differential diagnosis included Streptococcus pneumoniae, Legionella spp., Pasteurella spp., Hemophilus influenza, measles, simian varicella infection, and cytomegalovirus infection. Measles and simian varicella were considered unlikely because of the lack of cutaneous lesions. Aerobic bacterial culture of lung from this animal, and one other animal dying of pneumonia, revealed Bordetella bronchiseptica. The appearance of a “honeycomb” exudate within alveoli in some regions of the lung suggested the presence of Pneumocystis carinii, however, Gomori’s methenamine silver staining did not reveal organisms. Mycobacterium avium complex was also cultured from the lung of another affected animal at necropsy, but acid-fast staining of lung was unproductive and intradermal tuberculin testing of survivors was negative in all cases. A few animals from the group had detectable antibodies to measles virus, but rising titers were not found among survivors.
Other lesions in this animal included the following: mild, chronic, locally extensive, superficial tracheitis with squamous metaplasia; diffuse, severe, acute renal cortical tubular necrosis; focal acute necrosis of the adrenal cortex and adenohypophysis; diffuse moderate lymphoid depletion of the spleen; and locally extensive subcutaneous xanthomas of the foot pads.
Bordetella bronchiseptica has been reported as a cause of bronchopneumonia in African green monkeys and immunosuppressed human beings. These animals were not immunosuppressed. It may be that Bordetella bronchiseptica is especially pathogenic for African green monkeys. Bordetella bronchiseptica is a common commensal and/or pathogen in many species. No other species in proximity to these animals showed illness; animals housed in different rooms in the same building included dogs, swine, rabbits, mice and rats.
AFIP Diagnosis: Lung: Bronchopneumonia, necrohemorrhagic and fibrinosuppurative, acute, multifocal, severe, with cilia-associated coccobacilli, African green monkey (Cercopithecus aethiops), nonhuman primate.
Conference Comment: Bordetella bronchiseptica is a small, gram negative coccobacillus that is capable of producing infection in a wide variety of mammals. Dogs, pigs and guinea pigs are considered highly susceptible; rats, rabbits and horses have intermediate susceptibility; chickens, sheep, mice and man are least susceptible.
Normally, Bordetella bronchiseptica is an upper respiratory pathogen. The bacteria adhere to ciliated respiratory epithelium, replicate, cause ciliostasis, and host cell necrosis. In severe infections, such as this case, the bacteria involve the lower respiratory tract airways and may incite pulmonary damage. Usually, this is a result of infection with some other agent leading to decreased pulmonary defenses. Although Bordetella is capable of causing direct host damage, the inflammatory response and other concomitant infectious agents cause most of the lesions. Most of the cases reported in humans occurred in immunosuppressed persons.
Conference participants agreed that attempting to eliminate the possibility of underlying infection by agents such as simian retrovirus, simian immunodeficiency virus, morbillivirus, cytomegalovirus, adenovirus and parainfluenza virus was appropriate.
Contributor: Wake Forest University School of Medicine, Department of Pathology, Section on Comparative Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1040
References: 1. Dworkin MS, Sullivan PS, Buskin SE, Harrington RD, Olliffe J, MacArthur RD, Lopez CE: Bordetella bronchiseptica infection in human immunodeficiency virus-infected patients. Clin Inf Dis 28:1095-1099, 1999
2. Graves IL: Bordetella bronchiseptica isolated from
a fatal case of bronchopneumonia in an African green monkey. Lab
Anim Care 18(3):405-406, 1968
*Sponsored by the American Veterinary Medical Association, the American College of Veterinary Pathologists and the C. L. Davis Foundation.
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