Sabouraudia (1976), 14, 281-286

PROTOTHECOSIS

IN A CAT: FIRST RECORDED

CASE

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W. KAPLAN*, F. W. CHANDLER*, E. A. HOLZINGER~, R. E. PLUE~ AND R. 0. DICKINSON, III++

*Bureau of Laboratories, Centerfor Disease Control, Public Health Service, U.S. Department of Health, Education and Welfare, Atlanta, Georgia 30333, "~Diagnostic Assistance Laboratory, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, and +Chattahoochee Animal Clinic, RosweU, Georgia 30057 A 12-year-old Domestic Shorthair cat with a soft, fluctuant, subcutaneous mass, approximately 5 em in diameter on the posterior aspect of the left tarsus was diagnosed as having protothecosis. Cultures, histopathology, and fluorescent antibody reagents were usecL to identify Protothecawickerhamiias the etiologic agent. Protothecosis has not previously been recorded in cats. Members of the genus Prototheca are achlorophyllous microorganisms with a life cycle morphologically similar to that of species of algae in the genus Chlorella. Prototheca species characteristically produce hyaline cells, termed sporangia, which when m a t u r e divide by irregular cleavage to form 2-20 or more endospores. After sporulation is completed the sporangial wall ruptures and the released spores enlarge and repeat the reproductive cycIe (Arnold & Ahearn, 1972). T h e phylogeny and taxonomic position of the Prototheca species are in dispute. Some workers regard these organisms as achloric mutants of the green alga, Chlorella, on the basis of morphology and mode of reproduction (Cooke, 1968; Sudman, 1974). Others consider them to be fungi, because they are achlorophyllous and heterotrophic (Ciferri, 1957). Until recently, four Prototheca species, P. filamenta, P. stagnora, P. wickerhamii, and P. zopfii, were considered to be valid (Sudman, 1974). I n 1975 King & J o n g transferred P. filamenta to a new genus Sarcinosporon and n a m e d it S. inkin. Prototheca species can be identified by means of carbohydrate and alcohol assimilation tests (Arnold & Ahearn, 1972) and also by immunofluoreseence (Sudman & Kaplan, 1973). Prototheca species occur as saprophytes in the environment and have been recovered from such diverse sites as slime flux of trees, acidic stream and lake waters, marine waters, and sludges in waste stabilization ponds (Arnold & Ahearn, 1972). P. wickerhamii and P. zopfii under certain conditions have caused disease in m a n and lower animals (Buyukmihci, Rubin & DePaoli, 1975; Sudman, 1974; Sudman, Majka & Kaplan, 1973). While most lower animal cases involved internal organs, skin, regional lymph nodes, and eyes have been effected. Protothecosis has not been described previously in cats. This report documents protothecosis in a house cat caused by P. wickerhamii. CASE HISTORY A 12-year-old castrated male Domestic Shorthair cat, in generally good condition, was first examined on February 2, 1975. A soft, fluctuant, subcutaneous mass, approximately 5 cm in diameter, on the posterior aspect of the left tarsus was observed. The owner, first noticing the mass on December 14, 1974, was unaware of any antecedent injury at the lesion site. T h e cat having always resided in Georgia, and at its 281

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current residence for the past 7 years, had been permitted to r o a m out-of-doors at will; however, it generally returned home each night. A portion of the mass was excised, fixed in formalin for histological examination, and the operative site was cauterized with Lugol's solution. Because the lesion involved regional blood vessels and nerves, it could not be completely removed. The cat was given 125 mg of streptomycin and 100,000 units of procaine penicillin G* intramuscularly once a day for 3 days and then given a preparation containing tetracycline hydrochloride, novobiocin, and prednisoloneJ" orally for two days. T h e operative site healed uneventfully. By J u n e 1 t, 1975, the mass reappeared at the same site; a biopsy was taken for histological examination. T h e size of the mass was not measured at this time. O n August 5, 1975, the lesion was approximately 2-5 x 3-0 cm (fig. 1). A third biopsy was taken for cultural and histological examinations. The cat did not have any additional detectable cutaneous or subcutaneous lesions, and generally appeared to be in good health.

METHODS AND RESULTS

Gross Pathologic Findings There was no evidence of external skin involvement other than slight alopecia over the area of swelling. When surgically exposed, the subcutis contained a soft, uniformly grayish-white, glistening mass that blended imperceptibly with the surrounding tissues and could not be completely excised. The mass involved arteries, veins, and nerves. Areas of hemorrhage or necrosis were not evident. All three biopsies were grossly identical.

Histopathologic Findings Portions of the subcutaneous lesion were fixed in 10 percent neutral buffered formalin, embedded in paraffin, sectioned at 5 #m, and stained with hematoxylin and eosin (H&E), Gridley's method for fungi, and Gomori methenamine-silver with H & E counterstain. All three biopsies were histologically identical. T h e entire lesion consisted of granulomatous inflammation and m a n y spherical, oval, and crescent-shaped, nonbudding organisms in various stages of reproduction (fig. 2). M a n y organisms were observed within densely packed epithelioid and occasional foreign-body or Langhan's-type giant cells (fig. 3). Granulomatous tissue, with a b u n d a n t blood vessels, was supported by thin trabeculae of fibrous connective tissue. Multifocal areas of lymphocyte and plasma cell infiltration were seen, usually in perivascular locations (fig. 4). A few small foci of hemorrhage and necrosis were also present. Organisms were not found within blood vessels. Organisms ranged in size from 2-20 p m diameter. Reproductive stages included small, single undifferentiated forms; intermediate forms showing nuclear and cytoplasmic cleavage; and very large mature forms undergoing endosporulation containing 2 to 8 or more endospores. Most organisms were stained poorly or not at all by H & E. Inner structures were readily discerned in sections stained by Gridley's method or by the G o m o r i - H & E stain. T h e latter stain was ideal for photographic * Azimyc~'~a,Schering Corporation, Kenilworth, NewJersey. t Delta Albaplex, the Upjohn Company, Kalamazoo, Michigan.

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FELINE PROTOTHECOSIS

283

Figure 1 .--Soft, fluctuant subcutaneous mass on p l a n t a r surface of left tarsus (arrow) of a 12-yearold Domestic Shorthair cat. P h o t o g r a p h taken at third biopsy.

Figure 2.

D

Prototheca wickerhamii cells in subcutaneous tissue of left tarsus. Note undifferentiated a n d characteristic m a t u r e cells containing several endospores (arrows). GomoriH & E stain; x 1,125.

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Figure 3.--Aggregates ofProtothecawlckerhamiiwiehinforelgn-bodygiant ceilsand also extracellular. Note endosporulationin someceils. Gridley stain; x 500.

purposes since it demonstrated structural detail of the organisms and associated tissue changes. On the basis of size and morphology, the organisms were presumptively identified as a Prototheca species. Sections of tissue were digested in 1 ~ trypsin (Kaplan & Kraft, t969) and tested with species-specific fluorescent antibody (FA) reagents for the recognized Prototheca species (Sudman & Kaplan, 1973). The staining results confirmed the organism as P. wickerhamii. Cultural Findings

Tissue was cultured on Sabouraud dextrose agar containing chloramphenicol (0-05 mg/ml) and incubated at room temperature. Moist, creamy white, yeast-like growth contained cells with microscopic morphology compatible with protothecae. T h e isolate assimilated trehalose but not sucrose, inositol or n-propanol (Arnold & Ahearn, 1972) corroborating the results of FA staining.

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FELINE PROTOTHECOSIS

285

Figure 4.--Mild perivascular accumulation of lymphocytes and plasma cells in granulomatous tissue from subcutis. Most protothecae do no~ stain with H&E (arrow). It&E; x 500. DISCUSSION Protothecosis has many diverse clinical manifestations, depending upon the tissues which are affected. The signs and symptoms are not pathognomonic; laboratory studies are required for a definitive diagnosis. In this case, the disease appeared clinically as a localized, soft, fluctuant, subcutaneous mass closely resembling a chronic bite-wound abscess or a neoplasm. As in previously reported cases, a clinical diagnosis was not possible. To date, chemotherapy of protothecosis has not been effective. A variety of medicaments have been used with little success (Sudman, 1974). I n the present case, treatment with antibiotics and prednisolone was not specifically directed against the protothecal infection. Rather, these drugs were used to control secondary bacterial infection following surgery. It is apparent, however, that they did not cure the disease. Complete surgical excision of lesions has been an effective method of treating protothecosis in man (Rippon, 1974). Regrettably, in this case, only partial resection was feasible. Complete surgical excision of infected tissue could not be carried out due to the possibility of extensive damage to regional nerves and blood vessels.

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T h e c o m m o n o c c u r r e n c e o f p r o t o t h e c a e in t h e e n v i r o n m e n t suggests t h a t m a n a n d a n i m a l s m a y f r e q u e n t l y b e e x p o s e d to t h e s e o r g a n i s m s . H o w e v e r , few cases o f p r o t o thecosis h a v e b e e n r e p o r t e d . T h i s p a u c i t y c o u l d r e f l e c t m i n i m a l p a t h o g e n i c i t y of t h e s e o r g a n i s m s , or p o s s i b l y i n a b i l i t y to r e c o g n i z e o t h e r cases. G r e a t e r a w a r e n e s s o f this d i s o r d e r a n d i n c r e a s e d use o f a p p r o p r i a t e histological, i m m u n o l o g i c a l a n d c u l t u r a l d i a g n o s t i c p r o c e d u r e s , c o u l d y i e l d m o r e d i a g n o s e d cases. W e e m p h a s i z e t h a t t h e l a b o r a t o r y d i a g n o s i s o f p r o t o t h e c o s i s is n o t difficult. P r o t o t h e c a e a r e g e n e r a l l y a b u n d a n t in a f f e c t e d tissue, c a n b e r e a d i l y o b s e r v e d in tissue u s i n g s p e c i a l f u n g a l stains, h a v e a c h a r a c t e r i s t i c m o r p h o l o g y , a n d c a n be recovered on simple mycologic media. Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare. SOMMAIRE Un chat domestique de douze ans, avec une masse subcutan6, douce, fluctuant, approximativement 5 c m e n diametre sur l'aspect postfrieur de la tarsus gauche a 6t6 diagnostiqud d'avoir protothecosis. Cultures, histopathologie, et la technique de fluorescent anticorps ont dt6 usag6 pour identifier Prototheca wickerhamii comme l'agent etiologic. Protothecosis n'a pas au temps jadis, dt6 registr6 en chats. REFERENCES ARNOLD, P. & AHEARN, l). G. (1972). The systematics of the genus Prototheca with a description of a ne w species, P.filamenta. Mycologia, 64, 265-275. BIY'ZUKMIHCI,N., RUBIN, L. F. & DEPAoLt, A. (1975). Protothecosis with ocular involvement in a dog. Journal of the American Veterinary Medical Association, 167, 158-161. CIFERRI, O. (1957). Metabolismo delle Prototheca e delle mutallti achloride di Chlorella. Giornale di Microbiologia, 3, 97-108. CooxE, W. B. (1968). Studies in the genus Prototheca. I. Literature review. Journal of the Elisha Mitchell Science Society, 84, 213-216. KAPLA•, W. & KRAFT, DOROTHYE. (1969). Demonstration of pathogenic fungi in formalin-fixed tissue by immunofluorescence. American Journal of Clinical Pathology, 52, 420-432. KXNG, D. S. & JONG, S. C. (1975). Sarcinosporon: A new genus to accommodate Trichosporon inkin and Protothecafilamenta. Mycotaxon, 3~ 89-94. RIPPoN, J. W. (1974). Medical Mycology. The Pathogenic Fungi and the Pathogenic Actinomycetes p. 461. W. B. Saunders, Co., Philadelphia. gUnMAN,M. S. (t974). Protothecosis. A critical review. American Journal of Clinical Pathology, 61, 10-19. SUDMAN, M. S. & KA~LAN, W. (1973). Identification of the Prototheca species by immunofluorescence. Applied Microbiology, 25, 981-990. "S~JDMAN,M. S., MAJKA,J. A. & KAPLAN, W. (1973). Primary mucoeutaneous protothecosis in a dog. Journal of the American Veterinary Medical Association, 163, 1372-1374.

Protothecosis in a cat: first recorded case.

Sabouraudia (1976), 14, 281-286 PROTOTHECOSIS IN A CAT: FIRST RECORDED CASE Med Mycol Downloaded from informahealthcare.com by University Of Wisco...
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