Penicillium viridicatum mycotoxicosis in the rat. II. Scrotal lesions

Fd Cosmet.ToxicoLVol.IZ pp. 89-98.PergamonPress1974.Printedin Great Britain

PENICILLIUM

VIRIDICATUM

MYCOTOXICOSIS

II. S C R O T A L

IN THE

RAT.

LESIONS

M. D. McCRAcgEN and W. W. CARLTON Department of Veterinary Microbiology and Pathology, School of Veterinary Science and Medicine

and J. TUITE Department of Botany and Plant Pathology, School of Agriculture, Purdue University, West Lafayette, Indiana 47907, USA (Received 18 August 1973)

Abstract--Necrosis and ulceration of the scrotal epidermis developed in most rats fed cultures or fungal mats of Penicillium viridicatum for 3-6 wk. Advanced lesions were characterized by a severe necrotizing cellulitis of the epididymal adipose connective tissue and the scrotal fascia surrounding the tunicae and were accompanied by necrosis and ulceration of the epidermis. The progression of scrotal lesions was followed in a sequential study in which five rats were killed daily from day 3 to day 24 of the feeding of a 25~ fungal diet. The earliest changes occurred in the epididymal connective tissue as small focal accumulations of leucocytes, usually located perivascularly. Later, multiple foci of necrotic leucocytes accompanied by vasculitis of small arteries and veins appeared in the epididymal connective tissue. As the lesions increased in severity, loci of necrotic leucocytes developed in the connective tissue on the outside of the tunicae. Enlargement and coalescence of these foci produced lakes and bands of necrotizing cellulitis in the scrotal subcutis. Necrosis and ulceration of the epidermis occurred late in the sequence of changes. The same developmental pattern ofscrotal necrosis occurred in rats fed fungal mats. Extrascrotal skin lesions involving the back, tail and pelvic limbs were observed in rats fed fungal cultures for 6-8 wk. INTRODUCTION

Carlton & Tuite (1970) described gross lesions of the scrotum in rats fed a grain culture of Penicillium viridicatum. The microscopic changes consisted of a severe necrotizing cellulitis, which extended around the tunic sheaths and septa to the colon. Their studies indicated that the scrotal lesions were a direct response to mycotoxin(s) in the culture and suggested that lesions were induced by a blood-borne toxin. The lesions were studied after a certain period of feeding and as they were often advanced, the developmental stages were not defined. In the present study, an attempt was made to delineate the sequence of events in the development of scrotal lesions in rats fed fungal diets. EXPERIMENTAL

Experimental animals. Male rats were purchased at body weights between 250 and 350 g and were housed and fed as previously described (McCracken, Carlton & Tuite, 1974). Animals were examined daily and gross scrotal changes were investigated. Fungal diets. Rice cultures and fungal mats of P. viridicatum were prepared as previously described and mixed with a purified diet (McCracken et al. 1974). In Trial I, rats were fed rice cultures at dietary concentrations of 25 and 50% for 4--6 wk. Control rats were fed either purified diet or purified diet containing 35 or 50% autoclaved chloroformtreated rice. Trial II rats were fed a rice culture of P. oiridicatum at a dietary level of 25% 89

90

M. D. MCOtACKEN,W. W. CARLTONand J. Tut~ t

for 24 days, and five rats were killed by an overdose of ether each day from day 3 to day 24 of feeding. In Trial III, rats were fed fungal mats of P. oiridicatum at concentrations of 2-5 or 5"0~oin the diet. Autopsy. Gross alterations of the scrotum were recorded and the entire scrotum was removed and placed in 10~o formalin for fixation. Following fixation, 7-10 transverse sections were made through the scrotum starting 0"5-1.0 cm anterior to the prepuce and continuing to the posterior of the scrotum. The tissue was embedded in paraffin, sectioned at 6 nm and stained with haematoxylin and eosin. Samples of extrascrotal skin lesions were collected and processed similarly. RESULTS Trial I Gross scrotal lesions were first seen after the fungal diets had been fed for 2-3 wk. Initially, slight swelling about the prepuce extended distally over the scrotum for 1-2 cm. During the next 1-7 days, swelling progressively involved more of the scrotum, so that the anterior one-half, iwo-thirds or entire scrotum became involved (Fig. l). Swelling varied in severity ~imong the rats as did acuteness of onset. The scrotum was firm, while the skin was taut, thin and shiny, sometimes moist and usually either cyanotic of hyperaemic. This pattern of swelling occurred in approximately two-thirds of the test rats. Scrotal epidermal necrosis was seen after 18 days of feeding (Table 1). The longest period required for the development of scrotal necrosis was 31 days. Typically the initial focus appeared within 5-7 days after scrotal swelling, as small dark areas 1-6 mm in diameter on the midline approximately 1.0 cm posterior to the prepuce (Fig. 2). These lesions were round or elliptical, often with irregular edges, and were covered by a thin reddish-brown scab which later became thick and brownish-black. Usually within 12-48 hr, the focus of necrosis increased in size to give elliptical or oval areas from 1.0 to 1"5 cm in diameter (Fig. 3). At this time, secondary foci developed laterally or posteriorly to the initial lesion and increased in size during the next 1-2 days. Both primary and secondary foci expanded and coalesced until almost the entire scrotal epidermis was necrotic (Fig. 4). In rats with advanced lesions, only anterior-lateral and extreme posterior midline portions of the scroturn were uninvolved. In those that developed very extensive swelling, necrosis began as a reddish-brown discoloration of the skin which spread rapidly over the scrotum. The mildest histopathological alterations in the epidermis consisted of acanthosis and ballooning degeneration. More commonly, the changes included foci of necrosis, which in some rats was extensive, and ulceration of the epidermis. Ulceration was severe in some rats and craters extended deep into the scrotal fascia and blended with areas of necrotizing cellulitis (Fig. 5). In the scrotal fascia, a severe necrotizing cellulitis consisted of bands and focal ovoid accumulations of necrotic exudate extending from the dermis to the cremaster muscle and often completely circumscribing the scrotum. The scrotal fascia, collagen, blood vessels, muscle and leucocytes were necrotic as well. The exudate consisted of a mixture of inflammatory cells consisting of polymorphonuclear neutrophils, lymphocytes, plasma cells and macrophages. Infiltrates adjacent to areas of necrosis often consisted predominently of neutrophils. Small focal accumulations of lymphocytes often surrounded blood vessels within muscles and tunicae. The spaces between the tunicae surrounding the testes and the epidermis were widened by oedema and inflammatory cellular exudate. Vessels within

20 25 30

Experimental group

Purified diet* 25'~,. PVt 50~,, PVt

0 24 25

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Necrosis

Gross lesions

0 20 28

Necrotizing cellulitis of scrotum 0 17 24

Epidermal necrosis

0 20~ 21~

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No. of animals examined

Microscopic lesions

No. of organs affected

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In testes

Table I. Lesions of the scrotum and the male reproductire organs in rats fed a culture ofP. viridicatum (Trial I)

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the areas of cellulitis were often necrotic but some contained morphologically normal erythrocytes. Thrombosis involved vessels in areas undergoing necrotizing cellulitis and the walls of thrombosed vessels were focally or completely necrotic. Lesions of the testes were observed in about one-third of the test rats. Mild changes consisted of oedema and focal infiltration of mononuclear cells, but about one-third of the animals had small foci of necrotic tubules. In most of the rats with severe scrotal lesions, spermatogenesis was not affected. The penis was frequently involved and small areas of necrosis occurred ventral and lateral to the penile urethra. In a few animals, lesions were more severe and consisted of necrosis and bands of inflammatory exudate surrounding the urethra. No significant lesions of the scrotum or male reproductive tract were observed in control animals. No lesions were noted in the prostrate gland or seminal vesicles in either control groups or those fed fungal diets. Necrosis of the skin in sites other than the scrotum was observed in some test rats and involved the back, base of the tail, the tail and pelvic limbs. Lesions were usually 1-02.0 cm in diameter and appeared as well circumscribed thick brown scabs that were irregulax in shape: Some became ulcerated, with loss of the scab. The lesions of the legs and tail often circumscribed the organ. Trial II

Gross scrotal lesions were first observed in a few animals after 11 days of feeding as either a slight swelling about the prepuce or a slight generalized swelling of the scrotum. In a few rats, slight swelling of the anterior one-third to one-half of the scrotum was observed after 11-14 days of feeding. One-third of the rats killed daily from day 11 to day 21 had no gross lesions (Table 2) and the other rats killed from day 11 to day 18 had only slight scrotal swelling. Of the rats killed between days 20 and 24, 70% had obvious scrotal oedema and moderate extensive generalized swelling of the scrotum (Table 2). The scrotum was firm, sometimes reddened and moist, and had a few small erosions of the epidermis. Table 2. Gross serotal lesions in rats fed a diet containing 25% of a culture of P. viridicatum (Trial II) No. of rats with Gross swelling

Microscopic lesions

Day

No gross lesions

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Moderate

Marked

Mild

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0 0 0 0 0 0 3 2 3 I l I 0 0 0

0 0 0 0 0 0 0 0 0 I I I 3 0 3

Fig. I. Swelling of the s c r o t u m in a rat fed a rice culture o f P. t'h'idicatum. Depressions in the s c r o t u m are due to pressure on the organ as it rested on the b o t t o m of the cage. Fig. 2. T w o small areas of necrosis present distal to the prepuce near the midline o f the s c r o t u m of a rat fed a rice culture of P. t,iridicattm:. Fig. 3. Area o f ulceration revealed by the sloughing o f a scab o f the primary lesion. Secondary foci of necrosis are present posterior to and lateral to the primary lesion. Fig 4. An advanced lesion in which the necrotic loci have expanded and coalesced to involve m u c h o f the scrotal epidermis. Time between Figs 2 and 4 was approximately 4 days.

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Fig. 8 Area ofcellulitis in epididymal adipose connective tissue, containing an inflamed small artery (single arrow) and a necrotic venule (double arrow). H/E :.: 140. Fig. 9. Foci and bands of necrotic leucocytes in the epididymal adipose connective tissue of a rat fed a diet containing 2 5 ~ P. vh'idicattm~ culture. H/E ~-: 35.

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:ig. 13. Necrosis and ulceration of the epidermis and multiple foci of necrotic leucocytes in he scrotal subcntis of a rat fed 25 °~ P. z'h'i¢licatttm culture in the diet. H/E -. 56.

:ig. 12. Necrotizing cellulitis involving epididymal connective tissue and connective tissue ~utside tile tunica. Spermatozoa are present within the epididymal and seminiferous tubules tdjacent to areas of necrosis. H/E . 35.

:ig. 15. Cellulitis o f the scrotal fascia a b o u t the testicular tunicae with involvement of the crenaster muscle. H/E 56.

:ig. 14, Cellulitis of the scrotal subcutis in a rat fed fungal mats o f P . riridicalmn. H / E . 56.

Penicillium viridicatum myeotoxieosis

93

Histologically, scrotal changes were first observed at day 12 and consisted of congestion of vessels and small focal accumulations of inflammatory cells in the epididymal adipose tissue (Fig. 6). The cells of the exudate were predominantly mononuclear, consisting mainly of macrophages and lymphocytes, and were usually perivascular. As the lesions progressed in severity, most rats had a few or numerous foci of leucocytes or a diffuse infiltration of a mixture of inflammatory cells into the epididymal connective tissue (Fig. 7). The foci of leucocytes increased in size and some leucocytes were necrotic. These changes were accompanied by vascular lesions which were both inflammatory and degenerative and which affected both arterioles and venules (Fig. 8). Their walls contained mononuclear and polymorphonuclear inflammatory cells that also surrounded the vessel. Affected vessels were undergoing degeneration and the walls were palely acidophilic and finely granular. Some veins were dilated, their walls were pale staining and the lumina contained inflammatory cells. By days 16-18, some rats had small multiple foci of leucocytes outside the testicular tunicae. These foci were particularly numerous along the connective-tissue septa surrounding the tunicae and extending dorsally and laterally in the scrotal fascia. Inflammatory cells also occurred at the junction of the subcutaneous tissue with the scrotal adipose tissue and within the scrotal fat Later in the development, medium to large foci of mainly mononuclear inflammatory cells occurred within the epididymal fat (Fig. 9). The number of loci of moderate size outside the tunicae was increased and some of these contained necrotic cells. The principle site of involvement was the anterior epididymal adipose connective tissue, which contained both small and large foci of leucocytes undergoing necrosis. The adipose tissue was diffusely infiltrated in some rats by a mixture of inflammatory cells, which were predominantly mononuclear consisting of lymphocytes and macrophages. Neutrophils were present in small to moderate numbers in some foci and in the lumina of vessels and were occasionally scattered through the adipose tissue. Inflammatory cells were arranged as focal accumulations especially along the borders of the adipose tissue, in a linear arrangement along the capillaries, and as a diffuse infiltration of the epididymal adipose tissue. In animals with advanced lesions, changes were widely distributed. They occurred in the anterior epididymal fat and along the connective-tissue septa surrounding the cremaster muscle, and extended dorsally and laterally within the scrotal fascia. Within the anterior epididymal adipose tissue, variously sized foci mainly of mononuclear leucocytes involved major portions of the epididymal fat. These consisted of irregular foci, trabeculae and bands of leucocytes that were necrotic (Fig. 10). Fat necrosis was present, as was vasculitis, thrombosis and necrosis of vessels within the epididymal fat In rats with extensive gross swelling of the scrotum, the connective tissue between the cremaster muscle and the dermis was thickened by oedema, fluid and the lymphatics were markedly dilated (Fig. 11). Changes present outside the tunicae and in the loose connective tissue between the cremaster muscle and the dermis consisted of large foci and basophilic bands of necrotic leucocytes (Fig. 12), which followed the connective-tissue septa surrounding the cremaster muscle and extended dorsally and laterally within the scrotum. Large foci of necrotic leucocytes were observed in the cremaster muscle in approximately half the rats with advanced lesions. The inflammatory exudate involved the entire width of the muscle and extended into the loose connective tissue and fat on the outside of the muscle. Milder lesions of the cremaster muscle consisted of an infiltration of a mixture of inflammatory cells in the interstitium usually located perivascularly.

94

M.D. McCRACKEN.W. W. CARLTONand J. TUlXE

Necrosis and ulceration of the epidermis occurred in rats with advanced lesions (Fig. 13). In many rats the dermis and epidermis were unaffected, although severe necrotizing cellulitis was present in the scrotal subcutis. In some necrotic areas, inflammatory cells infiltrated the basal layers of the epidermis, and ballooning degeneration involved the epithelial cells adjacent to the areas of necrosis. In the papillary dermis beneath the necrotic areas, the mild cellular infiltration consisted mainly of neutrophils. Slight hyperplasia of the epithelium was observed in some areas. TrMl I I I

Slight scrotal swelling was observed in only two of the rats fed the P. viridicatum fungal mats at a concentration of 5-0~ of the diet, but this group was killed after 2 wk of feeding as the diet was highly toxic. Scrotal lesions occurred in nine of the ten rats fed the P. oiridicatum fungal mats at a concentration of 2"5~ in the diet. Slight scrotal swelling was seen after 2 wk of feeding in five rats. The changes gradually progressed in severity over the next 2 wk and in most rats the scrotum became obviously swollen, scaley and erythematous. Erosions, 2-3 mm in diameter, were observed in eight of the ten rats. The erosions were covered by a thin, dry, reddish-brown exudate, which persisted for several days and gl'adually healed, leaving a small scar on the surface of the scrotum. Microscopically, scrotal lesions were observed in eight of the ten rats fed fungal mats at a concentration of 5.0~ of the diet. In five rats the lesions were mild and consisted of focal accumulations of mononuclearceUs and vasculitis in the epididymal adipose connective tissue. In three, however, extensive cellulitis in the epididymal adipose connective tissue was characterized by large foci and bands of necrotic leucocytes and by small to moderately-sized foci of necrotic exudate in the scrotal fascia, between the tunicae and in the dermis (Fig. 14). Thrombotic vessels were observed in the epididymal connective tissue and outside the tunicae. Scrotal lesions occurred in all rats fed the fungal mats at a concentration of 2"5~ of the diet aod were usually most severe in the epidid),mal adipose connective tissue, where they occurred as variably sized foci of necrotic leucocytes. The most severe changes consisted of large areas of necrotizing cellulitis of the epididymal connective tissue and large bands of necrotic leucocytes in the scrotal fascia surrounding the tunicae (Fig. 15). In some rats, the connective tissue outside the tunicae was oedematous, blood vessels were congested and the lymphatic vessels were dilated. Necrosis and ulceration of the scrotal epidermis was present in eight of the ten rats. In most rats, the areas of necrosis and ulceration were small. Epidermal alterations included acanthosis and focal areas of hyperkeratosis and parakeratosis. Small focal areas of necrosis and infiltration of lymphocytes were present in the testes of two rats adjacent to the tunica albuginea. Degenerative changes in three rats consisted of a loss of spermatocytes, spermatids and spermatozoa. No morphological changes were present in the adjacent tubules. Penile lesions in two rats consisted of an accumulation of necrotic leucocytes ventral to the urethra.

DISCUSSION The scrotal lesions appeared to develop in response to one or more mycotoxins present in the culture and fungal mats of P. viridicatum. Scrotal necrosis does not occur in rats as an ageing or spontaneous change and no similar lesions have been reported in any spe-

Peniclllium viridicatura myeotoxieosis

95

cies of animal as-the result of an infection or mycotoxicosis. Budiarso, Carlton & Tuite (1970) described testicular lesions in mice fed the same isolate ofP. viridicatum, but a lesion of the scrotum was not found. The lesions began and were most numerous and severe within the epididymal fat. Infiltration of predominantly mononuclear cells adjacent to capillaries was followed by discrete foci of leucocytes, which increased progressively in size, and by vascular lesions. Focal leucocytic accumulations arose from multiple sites associated with vessels. Foci outside the tunicae tended to appear at junctions of adipose ceils and connective-tissue septa and larger bands and foci tended to follow connective-tissue septa within the scrotum. The cremaster muscle, which separates the testes and epididymis from the scrotal fascia, did not appear to play a significant role in the development of the lesions. Most cellular accumulations were focal and confined within the cremaster muscle, and severe lesions involving the muscle occurred only in rats with advanced lesions. The epidermis became involved at a late stage. Some rats'with severe lesions of the scrotal fascia and dermis had no epidermal lesions and involvement of the epidermis appeared secondary either to alteration of the blood supply or to toxic products produced in the areas of cellulitis. Carlton & Tuite (1970), studying advanced lesions in the scrotum of rats fed a rice culture concluded that the scrotal lesions were the results of blood-borne mycotoxin(s). The multicentric pattern of the scrotal lesions and their association with blood vessels supports this hypothesis. The development of lesions deep within the scrotal tissue with no involvement of the epidermis also indicates that the changes were the result of some circulating toxin(s) and were not due to contamination of the skin by the toxic diets. A number of fungal extracts and mycotoxins were toxic to the skin when applied topically, but the necrosis of the skin in our rats fed cultures of P. viridicatum apparently developed secondarily to the necrotizing lesions of the subcutis. Trichothecenes produced by Fusarium tricinctum, including diacetoxyscirpenol, T-2 toxin and HT-2 toxin, were highly toxic to the skin of rats, mice and rabbits following topical application (Bamburg & Strong, 1971). Lesions following application of T-2 toxin consisted of extensive inflammation and necrosis of the dermis and epidermis with extension of inflammation into the subcutis (Marasas, Bamburg, Smalley, Strong, Ragland & Degurse, 1969). The skin lesions in our rats appeared to begin as multicentric f6ci of necrosis associated with vessels and later progressed to a necrotizing cellulitis, which was followed by necrosis and ulceration of the epidermis, a progression similar to that in the scrotum. That skin lesions were the result of the activity of some blood-borne toxin(s) and not of surface contamination is suggested by the observations that a hepatotoxic chloroform extract of this isolate of P. viridicamm was non-toxic to the skin of mice (Budiarso, 1970). However, because species differences have been observed in responses to the application of various extracts to the skin, this line of evidence is not conclusive. Thus, Joffe (1971) reported that extracts of a Fusarium species highly toxic to rabbit skin were not toxic to the skin of rats and mice. The question of how soon the toxin results in detectable morphological changes can be estimated from the data obtained in this study. During the first 8 days, dilation of veins and lymphatics, congestion of the pampiniform plexus and a few small focal accumulations of mononuclear leucocytes were observed and were very similar to the histological appearance of a large number of scrotal sections taken from control rats. By days 12-14, the number of cellular foci had increased and cuffs of inflammatory cells surrounding capilF,C.T.

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96

M.D. McCt~cKEN, W. W. CAm.TONand J. T u l ~

laries and venules were the earliest unequivocal morphological evidence of a toxic and inflammatory effect. The congestion of the pampiniform plexus and dilatation of veins and lymphatics were present at all of the developmental stages. That the pampiniform plexus becomes involved somewhere in the sequence of events was indicated by the marked accumulation o f leucocytes about vessels and within the connective tissue of the plexus in animals with moderate to severe lesions. Bands and large foci of necrotic leucocytes, partieulady within the epididymal fat, appeared to result from the coalescence of adjacent foci, and it appeared that the extensive cellulitis resulted from the enlargement and coaleSence of multiple focal accumulations of leucocytes. Scrotal lesions induced by the feeding of the fungal mats were similar to those observed in rats fed fungal cultures. Scrotal lesions were first observed and were more severe in the epididymal adipose tissue, and involvement of the scrotal epidermis appeared to occur secondarily to these changes. These findings were similar to those in the sequential study, when the morphological progression of scrotal lesions was followed in rats fed the rice culture at a dietary level of 2 5 ~ Necrotizing inflammation involving the testis was seen in some rats, particularly those fed fungal diets for a longer period of time. It seems likely that necrosis of the testis was due to an extension of the necrotizing cellulitis. The fungal toxin(s) appeared primarily to affect loose connective tissue, as the epithelium of the epidermis and the germinal epithelium of the seminiferous tubules were not primarily altered. Degenerative changes and necrosis of the epithelium of the seminiferous tubules was not observed, except when the tubules were involved in necrotizing cellulitis. Budiarso et al. (1970) described testicular lesions in mice fed cultures of P. viridicatum and exposed to sunlight. Changes included vacuolation of spematocytes, formation of syncytial giant cells in the seminiferous tubules and necrosis of the tubular epithelium. In some mice, spermatozoa were absent or were present in decreased numbers. These changes were not observed in mice fed the fungal culture and not exposed to sunlight. Lesions in the testes have been reported with a number of mycotoxicoses and were observed following administration of extracts of F. nivale, nivalenol (Ueno, Ueno, Iitol, Tsunoda, Enomoto & Ohtsubo, 1971) and neosolanil (Ishii, Sakai, Ueno, Tsunoda & Enomoto, 1971; Ueno, Ishii, Sakai, Kanaeda, Tsunoda, Tanaka & Enomoto, 1972). However, the necrotizing cellulitis of the scrotum appears to be unique to rats fed P. viridicatum. Scrotal lesions were not observed in guinea-pigs (Carlton & Tuite, 1970) or mice (Budiarso et al. 1970) fed cultures of the same isolate of P. oiridicatum. Krogh, & Hasselager (1968) did not report scrotal lesions in rats fed a culture of P. viridicatum known to produce citrinin and oxalic acid (Krogh, Hasselager & Friis, 1970). Moreover, scrotal lesions have not been described in other studies of mycotoxins, including aflatoxin (Wogan, 1965), ochratoxin (Purchase & Theron, 1968), rubratoxin (Wogan, Edwards & Newberne, 1971) and sterigmatoeystin (van der Watt & Purchase, 1970). The scrotal necrosis induced in rats by the fungal diets was similar to a disease in man known as Fournier's idiopathic gangrene of the scrotum (Edington & Gilles, 1969; Talarieo, 1970). The aetiology of the condition in man is unknown, although a number of bacteria, including Staphylococcus aureus, streptococci, clostridia, Proteus spp. and Escherichia coil have been isolated from diseased tissues (Talarico, 1970). As these organisms are common surface contaminants, it is likely that most were secondary invaders. In man, extensive scrotal swelling was followed by necrosis, with sloughing of the distal two-thirds of the scrotum to expose the testicles (Edington & Gilles, 1969). The same pattern of

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swelling, necrosis and sloughing ofscrotal skin occurred in rats, but the testes were not often involved. Acknowledgemen ts---This work, published as paper no. 5232 of the Agricultural Experiment Station, Purdue University, West Lafayette, was supported in part by National Institutes of Health Grant ESCA-00463, United States Department of Agriculture Cooperative Agreement 12-14-100-9091 (5 I), Market Quality Research Division, and by National Institutes of Health Postdoctoral Special Fellowship No. FO3-GM-50866. The authors are indebted to Mrs. Nancy Martin and Mrs~ M. Thwaits for the histological preparations and to Mr. S. Royer for photographic assistance.

REFERENCES Bamburg, J, R. & Strong, F. M. (1971). 12,13-Epoxytrichothccenes. In Microbial Toxins. A Comprehensive Treatise. Vol. VII. Algal and Fungal Toxins. Edited by S. Kadis, A. Ciegler and S. J. Ajl. p. 207. Academic Press, New York. Budiarso, I. T. (1970). Investigations of the Mycotoxicosis Induced in Animals by the Fungus Penicillium viridicaturn. Ph.D. thesis, Purdue University. Budiarso, I. T., Carlton, W. W. & Tuite, J. F. (1970). Phototoxic syndrome induced in mice by rice cultures of PeniciUium viridicatum and exposure to sunlight. Path. vet. 7, 531. Carlton, W. W. & .Tuite, J. (1970). Mycotoxicosis induced in guinea pigs and rats by corn cultures of PeniciUium viridicatum. Toxic. appl. Pharmac. 16, 345. Edington, G. M. & Gilles, H. M. (1969). Pathology in the Tropics. p. 544. The Williams and Wilkins Company, Baltimore. Ishii, K., Sakai, K., Ueno, Y., Tsunoda, H. & Enomoto, M. (197 I). Solaniol, a toxic metabolite of Fusarium solani. Appl. Microbiol. 22, 718. Joffe, A. Z. (1971). Alimentary toxic aleukia. In Microbial Toxins. A Comprehensive Treatise. Vol. VII. Algal and Fungal Toxins. Edited by S. Kadis, A. Ciegler and S. J. Ajl. p. 139. Academic Press, New York. Krogh, P. & Hasselager, E. (1968~ Studies on fungal nephrotoxicity. Yearbook Roy. Vet. Agric. Coll., Copenhagen, p. 198. Krogh, P., Hasselager, E. & Friis, P. (1970). Studies on fungal nephrotoxieity. II. Isolation of two nephrotoxic compounds from Penicillium viridicatum Westling: citrinin and oxalic acid. Acta path, microbiol, scand. 78, 401. Marasas, W. F. O., Bamburg, J. R., Smalley, E. B., Strong, F. M., Ragland, W. L. & Degurse, P. E. (1969). Toxic effects on trout, rats, and mice of T-2 toxin produced by the fungus Fusarium tricinctum (Cd.) Snyd. et Hans. Toxic. appl. Pharmac. 15, 471. McCracken, M. D., Carlton, W. W. & Tuite, J. (1974). Penicillium viridicatum mycotoxicosis in the rat. I. Ocular lesions. Fd Cosmet. Toxicol. 12, 79. Purchase, I. F. H. & Theron, J. J. (1968). The acute toxicity of ochratoxin A to rats. Fd Cosmet. Toxicol. 6, 479. Talarico, R. D. (1970). Fournier's gangrene. Mod. Treat. 7, 1049. Ueno, Y., Ishii, K., Sakai, K., Kanaeda, S., Tsunoda, H., Tanaka, T. & Enomoto, E. (1972). Toxicological approaches to the metabolites of Fusaria. IV. Microbiology survey on "bean-hulls" poisoning of horses. Jap. J. exp. Med. 42, 187. Ueno, Y., Ueno, I., Iitol, Y., Tsunoda, H., Enomoto, M. & Ohtsubo, K. (1971). Toxicological approaches to the metabolites of Fusaria. III. Acute toxicity of fusarenon-X. Jap. J. exp. Med. 41, 521. van der Watt, J. J. & Purchase, I. F. H. (1970). Subacute toxicity of sterigmatocystin to rats. S. Afr. reed. J. 44, 159. Wogan, G. N. (1965). Chemical nature and biological effects of the aflatoxins. Bact. Rev. 30, 460. Wogan, G. N., Edwards, G. S. & Newberne, P. M. (1971). Acute and chronic toxicity of rubratoxin B t. Toxic. appl. Pharmac. 19, 712.

Mycotoxicose par Penicillium viridicatum chez le rat--ll. L~sions du scrotum R~sumt---La plupart des rats auxquels on a fait consommer pendant 3/l 6 semaines des cultures ou des agglom~rats fongiques de Penicillium viridicatum ont prtsent~ de la n/~crose et de l'ulctration de l'~piderme du scrotum. Les 16sions prononctes 6taient caract~ristes par une cellulite n~crosante grave du tissu conjonctif adipeux de rtpididyme et du fascia scrotal entourant les tuniques; elles 6taient accompagn~es de ntcrose et d'ulctration de l'~piderme. Afin de suivre la progression des l~sions du scrotum chez des rats soumis/l un rtgime alimentaire comportant 25% de P. viridicatum, on a sacrifi6 quotidiennement, du 3c au 24c jour du rtgime, cinq de ces animaux. Los premieres alttrations se sont manifestoes dans le tissue conjonctif de rtpididyme, sous forme de petits foyers

98

M.D. McCRAcrd~q, W. W. CARLTON and J. TUITE d'accumulation de leucocytes, g6n~ralement a localisation p~rivasculaire. Des foyers multiples de leucocytes n6crotiques et de l'inflammation des petites art~res et des veines sont apparus plus tard dans ce m~me tissu conjonctif. A mesure que les 16sions s'aggravaient, des foyers de leucocytes n~crotiques se sont constitu~s dans le tissu conjonctif de la face ext~rieure des tuniques. L'extension et la coalescence de cos foyers ont abouti a la formation de lacs et de bandes de cellulite n~crosante dans le tissue sous-cutan6 du scrotum. La nbcrose et l'ulc~ration de l'6piderme se sont produites tard dans la s~quence d'alt~rations. Le m~me sch6ma de n~crose du scrotum a ~t~ observ~ chez les rats auxquels on faisait consommer des agglom~rats fongiques. Des l~sions cutan~es extrascrotales, int6ressant le dos, la queue et les membres post6rieurs, ont ~t~ observ~es chez des rats qui ont consomm~ des cultures fongiques pendant 6 a 8 semaines.

Penicillium-eiridicatum-Mycotoxicose bei d e r R a t t e . II. S c r o t a l e L i / s i o n e n Zaummeafassuag--Necrose und Ulceration der scrotalen Epidermis entwickelten sich bei den meisten Ratten, an die 3-6 Wochen lang Kulturen oder Pilzfilze yon Penicillium viridicatum verftittert wurden. Fortgeschrittene I~sionen waren dutch schwere nekrotisierende Cellulitis des epididymalen Fettbindegewebes und der die Tunicae umgebenden scrotalen Fascien charakterisiert und wurden yon Necrose und Ulceration der Epidermis begleitet. Das Fortschreiten der scrotalen I~sionen wurde mit einem Reihenversuch vcrfolgt, in dem yore 3. bis 24. Tag der Verfiitterung eines 25~o Pilze enthaltenden Futters ~glich fiinf Ratten get/Stet wurden. Die friihesten Ver~nderungen traten in dem epididymalen Bindegewebe als kleine locale Leucocytenansammlungen auf, die gewShnlich perivascui~ lokalisiert waren. Sp~ter traten in dem epididymalen Bindegewebe multiple Herde nekrotischer Leucocyten begleitet yon Vasculitis kleiner Arterien und Venen auf. Als die Schwere der L~sionen zunahm, entwickelten sich Herde nekrotischer Leucocyten in dem Bindegewebe auf der Aussenseite der Tunicae. Durch VergrSsserung und Zusammenwachsen dieser Herde entstanden Ausuferungen und Biinder yon nekrotisierendcr Cellulitis in der scrotalen Subcutis. Necrose und Ulceration der Epidermis traten sp~it in der Folg¢' der Anderungen auf. Das gleiche Entwicklungsschema scrotaler Necrose trat bei Ratten auf, an die Pilzfilze verFtittert wurden. Extrascrotale Hautl~sionen auf dem Riicken, am Schwanz und an den Hinterbeinen wurden bei Ratten beobachtet, an die 6-8 Wochen lang Pilzkulturen verftittert wurden.