Comparative Medicine Vol 50, No 3 Copyright 2000 June 2000 by the American Association for Laboratory Animal Science

Granulomatous in the Opossum (Didelphis virginiana) Associated with an Intracellular Fungal Agent

C. M. Almgren1* and D. E. McClure2 Background and Purpose: Two of nine female opossums (Didelphis virginiana) in a closed breeding colony were submit- ted for necropsy due to a history of poor reproductive performance in the absence of overt clinical disease. On histologic examination, marked granulomatous to pyogranulomatous pneumonia was identified in these animals. Methods: sections were stained with periodic acid-Schiff and Gomori’s methenamine silver nitrate. Results: Pulmonary lesions were characterized by large numbers of foamy macrophages within the alveoli and interstitium, prominent subpleural and peribronchiolar aggregates of histiocytes, and a few scattered lymphoid nodules. Numerous fungal organisms were evident within the cytoplasm of macrophages on impregnation of histo- logic sections with the aforementioned stains. Other inciting agents were not identified. A third opossum lacked pulmonary lesions, but had similar organisms within one auricular sebaceous gland/hair follicle without apparent reaction to the organisms. Conclusion: A fungal agent was associated with granulomatous pneumonia in the opossum, and comparison was made with endogenous lipid pneumonia previously described in opossums. These findings stress the importance of use of special stains and additional diagnostic techniques when prominent alveolar macrophage accumulation is present on histologic examination of the opossum lung.

The Virginia opossum (Didelphis virginiana) is a species of often associated with either pulmonary or endog- importance in biomedical research, and the unique characteris- enous lipid pneumonia. “Endogenous lipid pneumonia is an al- tics and use of this species have been detailed. Despite history veolar filling disorder reported frequently in rats, and less of heavy parasite burden, numerous naturally acquired dis- frequently in other species such as mice, cats and humans” (2). eases, and equivocal reports of poor reproductive performance Gross lesions appear as multiple, occasionally coalescing, sub- and survivability in captivity, research use of these animals con- pleural white foci. Histologic lesions consist of foamy macroph- tinues. The current research importance of these animals de- ages within and often distending alveoli, sometimes associated mands further identification and investigation of spontaneous with foreign body-type reaction and cleft formation. diseases and opportunistic in this species. Any dis- The severity of the lesions varied within the nineteen cases de- ease process that increases stress in the animal has great po- scribed by Brown (2). The exact cause of endogenous lipid pneu- tential for affecting research outcomes. In addition, close monia in the opossum has not been identified, although chronic contact with wild-caught animals presents a possible source of pulmonary irritation and pulmonary nematode infestation have exposure of animal caretakers and research investigators to a been considered (2). A single report of granulomatous pneumo- variety of zoonotic diseases, because many of the pathogens car- nia in a Brazilian gray short-tailed opossum (Monodelphis ried by the opossum may be easily transmitted to humans. As domestica) associated with the microorganism Actinomyces we continue to expand our use and exposure to this species, we odontolyticus (12) has been published. should continuously be aware of previously unreported disease We identified a possible unreported (to our knowledge) cause conditions and potentially newly emerging pathogens. of granulomatous pneumonia in the opossum in which the gross Reports of lesions in the opossum have iden- and histologic lesions are strikingly similar to the lesions previ- tified several conditions, including but not limited to: Mycobac- ously described for endogenous lipid pneumonia. We evaluated terium intracellulare (1), endogenous lipid pneumonia the lesions, descriptive morphology of the intracellular fungal (2), pulmonary adenomas (3, 4), pulmonary adenocarcinoma (5), agent, results of use of fluorescent antibody techniques, and pulmonary parasitism (3, 4, 6, 7–11), and numerous miscella- transmission electron micrographs of the fungal agent within neous cases of pneumonia and often associated pulmonary alveolar macrophages. with bacterial endocarditis and mostly of unknown cause (3, 4, 7). Causes of granulomatous pneumonia in the Virginia opossum Materials and Methods have largely not been identified. Granulomatous pneumonia is History: Wild-caught opossums were acquired from a com- mercial vendor in southern Florida to be used for breeding pur- Department of Veterinary Biosciences1 and Preventive Medicine,2 The Ohio State poses. Newly acquired animals were quarantined for a University, Columbus, OH. minimum of three to six weeks, subjected to repeated tests for Dr. McClure’s present address is Campus Veterinarian/Director of Animal Resources, University of California, Santa Barbara, Santa Barbara, CA. endoparasites by use of fecal flotation and Baermann tech- *Corresponding author niques and were treated with fenbendazole (25 to 50 mg/kg of 323 Vol 50, No 3 Comparative Medicine June 2000 body weight orally; Panacur®, Hoechst Roussel Vet, Warren, NJ) protocols (13). Several unstained slides of each case that had until two negative results of fecal tests were obtained. Animals evidence of alveolar histiocytosis with intracellular fungal or- with evidence of ectoparasites were additionally treated with ganisms were submitted to The Centers for Disease Control and Sevin dust or carbaryl-based sprays. Some animals were given Prevention (CDC), Center for Infectious Disease, Atlanta Geor- enrofloxacin (5 mg/kg; Baytril®, Bayer Corporation, Agricultural gia for fluorescent antibody evaluation. A 1-mm section of lung Division, Animal Health, Shawnee Mission, KS) for 7 to 10 days, identified as containing fungal organisms by evaluation of as deemed necessary for treatment of bacterial infection by the GMS-impregnated sections was trimmed from the tissue block, consulting veterinary staff. All animals were housed at the Ohio deparaffinized, further fixed in 3% glutaraldehyde and 1.33% State University Laboratory Animal Facility, Columbus Ohio. osmium tetroxide, and embedded in epoxy resin. Ultrathin sec- The facility is fully approved by the Association for Assessment tions (800 Å ) were prepared by use of an LKB Ultra-microtome and Accreditation of Laboratory Animal Care International and were stained with uranyl acetate and lead citrate. A (AAALAC). The animals were handled according to the Insti- Phillips 300 transmission electron microscope was used for vi- tute for Laboratory Animal Resources (ILAR) Guidelines for the sualization of the ultrathin sections. Care and Use of Laboratory Animals. After participation in an in- stitutional laboratory animal care and use committee (ILACUC)- Results approved breeding protocol, bodies of the animals were submitted Gross examination: All opossums were considered to be for pathologic examination. middle-aged sexually intact females in apparent good condition After the quarantine period, all breeding females were singly with adequate stores of body fat. In opossum 1, the were housed in indoor/outdoor kennel-type enclosures having cement moderately atelectatic, with numerous 1- to 5-mm yellowish white floors, concrete block walls separating individual enclosures, foci evident on the pleural surface and within the parenchyma. A and chain link fencing covering the top and doorway. The enclo- moderate amount of clear frothy fluid exuded from the cut surface sure was additionally fenced at the perimeter to prevent expo- of the lung (interpreted as ). Other lesions in this sure of these animals to indigenous wildlife species. Opossums animal consisted of a 1.2-cm red depressed focus on the plantar were the only species housed in this portion of the building. El- aspect of the hind foot interpreted as superficial ulceration; a 2-cm evated nest boxes lined with bedding were provided for breed- abscess on the ventral aspect of the tail base with a central drain- ing purposes. Females with pouch young were housed indoors in ing tract; onychitis affecting multiple digits, with loss of the nail stainless steel cages. Diet consisted of wet and dry dog food from two digits; numerous parasites, the gross appearance of supplemented with fruits, vegetables, and insects (principally which was consistent with Physaloptera sp. within the stomach; meal worms) provided fresh daily. Cleaning and cage mainte- and multiple firm to gritty yellow 0.5- to 1.5-cm nodules within the nance were performed as needed, but were kept to a minimum mesentery surrounding the stomach and pancreas. during breeding and gestation periods. The lungs of opossum 2 contained several 1.0- to 2.0-mm firm Females were periodically placed with individual males for 5- white nodules scattered throughout all lung lobes. Addi- day breeding periods, then were removed. The females in this tional lesions included moderate amount of dental tartar on the group arrived at our facility in March and were housed in this en- upper premolars and molars and a 1.5-cm ulcerated lesion on closure until October of the same year. These animals were culled the right hind foot. from the breeding group and euthanized because of a history of On external examination of opossum 3, a 2-mm slightly poor reproductive performance in the absence of other clinical raised dark black focus was present on the inner pinnae near signs. All animals were deeply anesthetized with sodium pentobar- the caudal aspect of the left ear. Onychitis was evident on the bital (Nembutal® sodium solution, Abbott Laboratories, North Chi- 5th digit of the left forefoot. A small, 2.0- to 3.0-mm, ulcerated cago, IL.) and were euthanized by intracardiac perfusion of lesion was present on the dorsal aspect of the tail, approxi- formalin. The brain and spinal cord were removed for other re- mately 3.0 mm from the tip of the tail. Gross lesions were not search purposes. The animals were then immediately transported evident in the lungs. The gastric lumen contained numerous to the pathology laboratory for complete necropsy. parasites, the appearance of which was consistent with Physa- Necropsy: Pathologic evaluation included thorough gross ex- loptera sp. Several 1.0- to 2.0-mm white foci were visible on the amination and collection of tissue specimens from all major or- cut surface of the spleen. A 4.5 x 3.0-cm soft yellowish red fri- gan systems (excluding the nervous system) and any lesions able mass was present in the right oviduct/uterine horn, ap- that were identified. All tissue specimens were fixed in neutral- proximately 2.0 cm distal to the ovary. buffered 10% formalin. Histologic examination: Lung lesions in opossum 1 con- Tissue processing: After adequate fixation, tissue speci- sisted of marked diffuse granulomatous to pyogranulomatous mens were trimmed and submitted to the histopathology labo- (Figure 1), moderate pulmonary edema, ratory for routine automated processing (Fisher Histomatic™ multifocal , and a few foci of osseous metaplasia. Ex- Tissue Processor Model 166A, Fisher Scientific Company, In- amination of tissue sections from opossum 2 revealed diffuse strument Division, Pittsburgh, PA), paraffin embedding, and moderate chronic granulomatous bronchointerstitial pneumo- sectioning. Tissue sections, 5-um thick, were affixed to glass nia with subpleural, perivascular, and peribronchiolar histio- slides, processed, and stained with hematoxylin and eosin (H&E). cytic and lymphoplasmacytic inflammation; lymphoid nodule Special stains, fluorescent antibody testing, and trans- formation (Figure 2); type-II pneumocyte hyperplasia; and mild mission electron microscopy: Specimens of lung with granu- smooth muscle hypertrophy/hyperplasia. A few foci of mild os- lomatous pneumonia were additionally subjected to Gomori’s seous metaplasia also were detected. In both of the aforemen- methenamine silver (GMS) nitrate method, periodic acid-Schiff tioned opossums, alveoli were filled and often distended by (PAS), Kinyoun’s acid-fast, and Gram staining as per standard numerous pulmonary alveolar (foamy) macrophages (Figure 3). 324 Granulomatous Pneumonia in the Opposum

Figure 3. Higher magnification of pulmonary parenchyma (opossum Figure 1. Photomicrograph of a section of the lung from opossum 1. 1). Notice filling of alveoli in the opossum lung with numerous large Notice dense cellular infiltration of the interstitium and most of the foamy macrophages. These cells are identified by their eosinophilic alveoli with pulmonary alveolar (foamy) macrophages. H&E stain; foamy to vacuolated cytoplasm (*) and large ovoid- to bean-shaped bar = 200 ␮m. nuclei (arrowhead). A few neutrophils also are present. H&E stain; bar = 15 ␮m. lymphoplasmacytic and neutrophilic hepatitis (random, portal, and periportal); multifocal mild neutrophilic tubulointerstitial ne- phritis; moderate to marked diffuse chronic lymphoplasmacytic endometritis; multifocal marked chronic necrotizing and ulcerative dermatitis of the tail and feet; marked periosteal new bone forma- tion associated with the tail lesion; severe chronic suppurative pododermatitis, osteomyelitis, cellulitis and arthritis of the digits; mild to moderate extramedullary hematopoiesis in the spleen and adrenal glands; focal minimal nodular adrenocortical hyperplasia; and multiple parasitic granulomas within the mesentery. Opossum 2 had multifocal eosinophilic gastritis associated with mineralized parasitic granulomas; chronic lymphoplasmacytic and eosinophilic interstitial pancreatitis with associated fat necro- sis and granuloma formation; minimal mesangioproliferative glomerulopathy; mild chronic neutrophilic and lymphocytic por- tal hepatitis with fibrosis, mild biliary hyperplasia, and multifo- cal periacinar lipidosis; widespread mild eosinophilic enteritis; mild multinodular adrenocortical hyperplasia; mild chronic lymphoplasmacytic endometritis and cervicitis; focal minimal suppurative oophoritis; and minimal to mild extramedullary hematopoiesis in the liver, spleen, and adrenal glands. Figure 2. Photomicrograph of a section of pleural and subpleural regions of lung from opossum 2. Notice marked infiltration with al- Nonpulmonary lesions of interest in opossum 3 included moder- veolar histiocytes, multifocal lymphoid nodule formation (arrows), and ate diffuse circumferential centrilobular hepatic fibrosis thought to multifocal mild to moderate smooth muscle hypertrophy/hyperplasia be consistent with veno-occlusive disease; diffuse marked chronic (arrowheads). H&E stain; bar = 300 ␮m. endometritis; benign dermal melanoma of the left ear; one auricu- lar sebaceous gland/hair follicle that contained fungal organ- The third animal did not have evidence of pneumonia or alveo- isms without apparent cellular reaction; eosinophilic and lar histiocytosis. Lung lesions in this animal consisted of mul- lymphoplasmacytic gastritis; focal chronic gastric ulceration; tifocal moderate to marked smooth muscle hypertrophy/ adenomatous hyperplasia of the right uterine horn; suppurative hyperplasia, multifocal mild emphysema, focal mild adenomatous pododermatitis, osteomyelitis and cellulitis of one digit; focal necro- hyperplasia, and prominent perivascular and peribronchiolar tizing and ulcerative dermatitis of the tail; and extramedullary lymphoid tissue. hematopoiesis in the liver, spleen, and adrenal glands. Other lesions of interest in opossum 1 included mild to moderate Special staining of lung sections: Numerous 2- to 6-␮m 325 Vol 50, No 3 Comparative Medicine June 2000

Figure 4. Serial section of lung (opossum 1) seen in Figure 3 pre- Figure 5. Transmission electron micrograph (TEM) of an alveolar pared by Gomori’s methenamine silver nitrate (GMS) impregnation macrophage from an opossum. Notice the nucleus (N) and numerous highlighting fungal organisms (identified by arrows) within the cyto- cross and longitudinal sections of fungal organisms (*), surrounded plasm of alveolar macrophages. Bar = 15 ␮m. by clear space, within the cytoplasm. Arrowheads indicate cell walls of the fungi, and an arrow points to a lipid vacuole within the cyto- oblong yeast-like organisms with evidence of budding were evi- plasm of one fungal organism. Bar = 0.8 ␮m. dent within pulmonary alveolar macrophages (Figure 4) on GMS (dark black organisms on a green background)- and peri- odic acid-Schiff (not shown)- stained lung sections. Tissue mor- phology of these fungal organisms varied from small oval, oblong to spherical yeast with narrow based budding and some organisms with cylindrical, cigar- or comma-shaped, elongated rod-type forms. The morphology of the yeast in tissue appeared similar to, but was more elongate, and had budding characteris- tics different from that of Sporothrix schenckii. Rhodotorula sp. also was considered, but again, the tissue morphology indicated some variation from this fungal species. The morphology of the yeast in question did not conform to that of any known common yeast form (14). Sections were additionally stained with Kinyoun’s acid-fast and Gram stains, and acid-fast, gram-posi- tive, or gram-negative microorganisms were not identified. Fluorescent antibody techniques: Fluorescent antibody testing of samples submitted to the CDC Division of Bacterial and Mycotic Disease yielded negative fluorescent antibody reac- tion to conjugates specific for S. schenckii (Y form) and Candida sp. A positive 2+ staining reaction to a bivalent Histoplasma capsulatum-Blastomycosis dermatitidis conjugate (Y form) was detected. We believe that this positive reaction was due to non- specific cross-reactivity, because the morphology of the organ- Figure 6. Higher magnification TEM of the fungal organisms within isms did not conform to the morphology of either H. capsulatum the cytoplasm of pulmonary alveolar macrophages (opossum lung). or B. dermatitidis. Two cross sections (*) of fungal organism are evident. Although the Electron microscopy: On transmission electron microscopy of organism at the upper left is slightly degenerate, the thick fungal cell ␮ wall (arrow), cytoplasmic vacuoles, lipid globules, and dense granules ultrathin sections, 2- to 6- m, single-celled organisms surrounded are evident. Internal structures/organelles, including the fungal cell by a peripheral clear zone were present within the cytoplasm of nucleus (N), a mitochondrion (M), and an intracytoplasmic vacuole alveolar macrophages. Often, numerous fungal organisms were (V), are labeled in the cross section at the lower right. Bar = 0.5 ␮m. present within each alveolar macrophage (Figure 5). They are identified as fungal organisms by presence of a thick outer cell wall Discussion and internal structures, including a nucleus, mitochondria, lipid Although granulomatous pneumonia is common in the North globules, cytoplasmic vacuoles, and free ribosomes (15) (Figure 6). American opossum, the etiopathogenesis, in most instances, re- 326 Granulomatous Pneumonia in the Opposum

mains unknown. Most cases of granulomatous pneumonia in and heart blood on Sabauraud’s dextrose agar with chloram- this species are attributed to chronic pulmonary parasitism or a phenicol and cycloheximide or brain heart infusion agar with condition described as endogenous lipid pneumonia, an alveolar blood and chloramphenicol/cycloheximide and incubation at 25 filling disorder. One report described granulomatous interstitial and 37ЊC are recommended (14). Unfortunately in these in- pneumonia associated with the “atypical” mycobacterium, stances, the animals had been perfused prior to examination by M. intracellulare, in two Virginia opossums (1). Actinomyces the pathologist and fresh lung tissue and blood were unavailable. odontolyticus has been identified as the agent involved in a The cases presented here represent a unique finding in the single case of granulomatous pneumonia in the grey short- Virginia opossum. Mycotic pneumonia and mycotic disease in tailed opossum (Monodelphis domestica) (12), but similar micro- the opossum, in particular, are rarely reported. A report of Tri- organisms have not been reported in the Didelphis spp. chophyton mentagrophytes and Microsporum sp., without the We have identified two cases of an intracellular fungal agent presence of visible dermal lesions, has been reported (16). Histo- associated with granulomatous pneumonia in D. virginiana. plasma capsulatum and B. dermatitidis also are known to affect The fungi are abundant; often numerous organisms are found this species, but again, reports are rare. One study (17) exam- within the cytoplasm of individual alveolar macrophages. Other ined a subpopulation of rats and an opossum in Detroit, Michi- than presence of the fungi and lack of visible cholesterol depos- gan, and revealed the presence of six fungal species from four its, the lesions in these cases are similar to those reported for genera in the opossum including: two Penicillium sp, two endogenous lipid pneumonia in this species. On gross examina- Trichothecium sp, one Cryptococcus sp, and T. mentagrophytes. tion, both conditions are associated with multifocal subpleural Trichophyton mentagrophytes was seen in association with white to yellowish white foci or stippling. Similarities in the his- small dermal lesions, and other lesions were not identified. topathologic findings include filling (often with distention) of It is uncertain whether the cases presented here represent a pulmonary alveoli with numerous foamy macrophages, promi- previously unreported or newly emerging pathogen of the opos- nent pleural and subpleural accumulations of alveolar histiocytes, sum lung. The possibility also exists that these animals might and often, sometimes prominent, lymphoid nodule formation. have been immunocompromised, but considering that they were The severity of the lesions varied with the sections of lung clinically normal and had not been subjected to previous experi- examined, even in an individual case. In some sections, the or- mental manipulation, this would seem an unlikely explanation ganisms were detectable on H&E-stained section, but sections for the lesions observed. Although the identity of the fungal or- taken from other regions of pulmonary parenchyma in these ganism remains unknown and it is still uncertain whether the cases would have been indistinguishable from those of endog- organism represents a primary pulmonary pathogen or second- enous lipid pneumonia without use of special staining tech- ary opportunistic invader, the fungal agent is associated with niques (GMS, PAS) to highlight the organisms. On electron considerable pulmonary disease that cannot be attributed to microscopy, the fungal organisms were easily distinguishable other etiologic agents on the basis of results of pathologic ex- from the osmophilic bodies seen within the cytoplasm of mac- amination. The similarities with endogenous lipid pneumonia rophages in cases of endogenous lipid pneumonia. are striking and warrant use of special stains for differential In opossum 3, similar fungal organisms were present within diagnosis when marked alveolar histiocytosis is present in the one auricular sebaceous gland/hair follicle without evidence of opossum lung. The severity of the pulmonary lesions in these cellular or tissue reaction and would have gone unnoticed if the cases may affect the overall general health, condition and stress tissue had not been processed to examine dermal melanoma. levels of the animals and has the potential to substantially in- There was no evidence of granulomatous pneumonia in this ani- fluence the results of some types of research. mal, and pulmonary lesions consisted only of smooth muscle As the impetus continues to find alternative species for use in hypertrophy/hyperplasia, adenomatous hyperplasia, and promi- biomedical research, the scientific community will experience nent perivascular and peribronchiolar lymphoid tissue. It is increased exposure to some “exotic” and wild animal species. In possible that these lesions may represent chronic pulmonary ir- many instances, these animals have not been thoroughly stud- ritation or previous pulmonary parasitism. It is interesting that ied with respect to spontaneous disease processes, and often in evidence of pulmonary parasites was not present in these three the case of wild animal species, newly emerging or previously animals. This may reflect previous treatment with a parasiti- unreported disease conditions exist. Although literature is rap- cide, but it would seem that some remaining evidence of previ- idly addressing problems in these species, much work remains ous pulmonary parasitism would have been identifiable to be done. The responsibility lies with the laboratory animal, microscopically, especially considering the fact that these ani- pathology, and research investigative staff to identify and report mals still had live Physaloptera sp. within the gastric lumen new or unusual conditions in these species. In today’s research and lesions within the stomach and mesentery that could be at- environment, many techniques, some fairly simple and others tributed to previous parasite infestation. quite sophisticated, are available to aid in diagnosis. These tech- Attempts to classify the yeast in these animals were unsuccess- niques may often aid in arriving at the etiopathogenesis of pre- ful. The organisms had structural similarities to S. schenckii and viously unreported disease conditions. Rhodotorula sp., but budding and other minor morphologic characteristics differed among these organisms. Morphology of the fungal agent identified in these cases did not match any of Acknowledgments the serotyped fungal pathogens. We thank Leo Kaufman and associates at the CDC Division of Bac- terial and Mycotic Disease Atlanta, GA for performing fluorescent Ideally, fresh specimens of lung tissue and heart blood should antibody testing, The Ohio State University Department of Veteri- be cultured, and serum should be obtained for fungal testing to nary Biosciences Pathology, Preventive Medicine and Biomedical aid in identification of fungal organisms. Culture of lung tissue Media departments for use of equipment and for expertise provided, 327 Vol 50, No 3 Comparative Medicine June 2000

Anne E. Handley for assistance with transmission electron micros- 8. Duncan, R. C., Jr., C. R. Reinemeyer, and R. S. Funk. 1989. copy and especially S. E. Weisbrode and Julie Hutt for review of the Fatal lungworm infection in an opossum. J. Wildl. Dis. 25(2): histopathologic and associated portions of the text. 266–269. 9. Nettles, V. F., A. K. Prestwood, and W. R. Davidson. 1975. Severe parasitism in an opossum. J. Wildl. Dis. 11(3):419–420. 10. Prestwood, A. K., V. F. Nettles, and R. L. Farrell. 1977. Patho- References logic manifestations of experimentally and naturally acquired 1. Moore, T. D., A. M. Allen, J. R. Ganaway, et al. 1971. A fatal lungworm infections in opossums. Am. J. Vet. Res. 38(4):529–532. infection in the opossum due to Mycobacterium intracellulare. 11. Snyder, D. E., A. N. Hamir, C. A. Hanlon, et al. 1991. Lung J. Infect. Dis. 123(6):569–578. lesions in an opossum (Didelphis virginiana) associated with 2. Brown, C. C. 1988. Endogenous lipid pneumonia in opossums Capillaria didelphis. J. Wildl. Dis. 27(1):175–177. from Louisiana. J. Wildl. Dis. 24(2):214–219. 12. Schenkman, D., F. Bertram, and R. Reynolds. 1994. Granu- 3. Sherwood, B. F., D. T. Rowlands, Jr., and D. B. Hackel. 1969. lomatous pneumonia in a Brazilian gray short-tailed opos- Pulmonary adenomatosis in opossums (Didelphis virginiana). sum (Monodelphis domestica) associated with Actinomyces J. Am. Vet. Med. Assoc. 155(7):1102–1107. odontolyticus. Vet. Pathol. 31(5):593 (abstr.) 4. Vakilzadeh, J., B. F. Sherwood, D. B. Hackel, et al. 1971. 13. Sheehan, D. C., and B. B. Hrapchak. 1980. Theory and prac- Experimental study of pulmonary adenomas in the opossum (Di- tice of histotechnology, 2nd ed. Batelle Press, Columbus, OH. delphis virginiana). Lab. Anim. Sci. 21(2):224–228. 14. Kaufman, L. 1998–99. Personal communication. 5. Barrie, M. T., and R. L. Snyder. 1986. Multiple primary neo- 15. Cheville, N. F. 1994. Ultrastructural pathology: an introduction plasms in an opossum. J. Am. Vet Med. Assoc. 189(9):1160–1161. to interpretation, first ed. Iowa State University Press, Ames, IA. 6. Flatt, R. E., L. R. Nelson, and N. M. Patton. 1971. Besnoitia 16. Potkay, S. 1970. Diseases of the opossum (Didelphis Marsupialis): darlingi in the opossum (Didelphis marsupialis). Lab. Anim. Sci. a review. Lab. Anim. Care. 20(3):502–511. 21(1):106–109. 17. Thiermann, A. B., and C. D. Jeffries. 1980. Opportunistic fungi 7. Sherwood, B. F., D. T. Rowlands, Jr., D. B. Hackel, et al. in Detroit’s rats and opossum. Mycopathologia 71(1):39–40 1969. The opossum, Didelphis virginiana, as a laboratory animal. Lab. Anim. Care. 19(4):494–499.

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