RESEARCH NOTES 253

•—, and J. Sarmiento. 1985. Observations on the Parenti, L. R. 1984. A taxonomic revision of the fishes of the Bolivian part of Lake Titicaca. II. Andean killifish genus Orestias, Cyprinodonti- Orestias agassii, Pisces, Cyprinodontidae. Revue formes, Cyprinodontidae. Bulletin of the Ameri- d'Hydrobiologie Tropicale 18:159-170. can Museum of Natural History 178:110-214.

J. Helminthol. Soc. Wash. 62(2), 1995, pp. 253-256

Research Note Histopathology of Oligacanthorhynchus tortuosa () Infection in the Virginia Opossum (Didelphis virginiand)

DENNIS J. RICHARDSON AND EARL B. BARN A WELL School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588-0118

ABSTRACT: Oligacanthorhynchus tortuosa, a common globular bears 6 spiral rows of 6 hooks acanthocephalan of the Virginia opossum (Didelphis each and has a length of 0.22-0.23 mm and width virginiana} in North America, has been reported to be associated with large, nodule-like lesions at points of of 0.23-0.29 mm (Van Cleave, 1953). Leidy attachment of the proboscides. Three lesions resulting (1850) reported a specimen of O. tortuosa as hav- from the attachment of individuals of O. tortuosa, 1 ing the anterior 3 lines of its length buried in an each from 3 infected opossums, were prepared for his- oval tumor, 4 lines in diameter, in the mesentery tological examination to further characterize histo- of an opossum. Based on this statement, Van pathologic changes elicited by this parasite. Histolog- ically, lesions involved the mucosa, submucosa, and Cleave (1924) concluded that the worm had pen- muscularis. The proboscides were contained within ab- etrated the intestinal wall, entered the body cav- scesses characterized by necrotic debris interspersed ity, and attached to the mesentery. Feldman et with many pycnotic nuclei. The abscesses were ap- al. (1972) reported severe ulcerative lesions proximately 1.4 mm in diameter and were surrounded by regions of dense connective tissue (collagen), ap- evoked at points of attachment of unidentified proximately 142 /um wide. The bands of dense con- acanthocephalans from opossums. Brief descrip- nective tissue were surrounded by regions of active tion and a photomicrograph (Feldman et al., fibroblast and fibrocyte proliferation, approximately 1972) suggest that these specimens were O. tor- 169 jum wide, in which evidence of collagen synthesis tuosa. Richardson et al. (1992) reported 2 poorly was observed. Both longitudinal and smooth muscle layers of the muscularis had been completely destroyed developed individuals of O. tortuosa from the in the area of the lesion. Absence of polymorphonu- small intestine of a (Procyon lotor) that clear leukocytes were indicative of chronic lesions. His- caused "severe lesions" at points of attachment; topathologic changes elicited by O. tortuosa include however, histological examination was not con- chronic inflammatory response to mechanical trauma resulting from injury caused by the proboscis with sub- ducted. The only histological examination of le- sequent fibrosis and nodule formation. sions caused by O. tortuosa was conducted by KEY WORDS: histopathology, Oligacanthorhynchus Babero (1957), who reported elevated nodules tortuosa, Didelphis virginiana, opossum, Acanthoce- over the serosal surface of the small intestines of phala. 2 Illinois opossums having a base diameter of 2-7 mm. He reported the nodules to have a bright Oligacanthorhynchus tortuosa, a common red appearance due to congestion of intestinal acanthocephalan of the Virginia opossum (Di- blood vessels. Histologically, lesions reported by delphis virginiand) in North America, has been Babero (1957) resulted in complete mechanical reported to be associated with large, nodule-like destruction of the mucosal and submucosal lay- lesions at points of attachment of proboscides. ers with some focal atrophy and necrosis of the Oligacanthorhynchus tortuosa is represented by muscularis. He further noted limited leukocytic large worms with females achieving lengths of infiltration and some pigment deposition. Ba- up to 350 mm (Richardson, unpubl. data). The bero (1960) examined opossums from Georgia

Copyright © 2011, The Helminthological Society of Washington 254 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY OF WASHINGTON, 62(2), JUL 1995 infected with O. tortuosa in which no such hem- orrhagic lesions were observed. The purpose of this investigation was to fur- ther characterize lesions resulting from O. tor- tuosa infections in the Virginia opossum. Three lesions resulting from the attachment of individuals of O. tortuosa, 1 each from 3 opos- sums, were prepared for histological examina- tion. Material was obtained from opossums col- lected in the course of a survey of Acanthoce- phala of opossums from Arkansas, results of which were reported by Richardson (1993). All lesions examined were caused by mature worms. '%''*'' ^^^&^3^£^& '*' Immediately after sacrificing the opossum, the '^ '"•':fcSr^te"^"'/.^s-^sS^' ' ^ small intestine was examined for nodules on the serosal surface, then removed and longitudinally i-^^P^^M ;£ v\-vr>^;:"- ~r$g. --;m dissected. Mature worms were severed so as to leave the proboscis intact and undisturbed in the lesion. The lesion along with normal tissue im- mediately surrounding the lesion was excised and placed directly into Bouin's fixative. After fixing in Bouin's solution for 24 hr, tis- sues were stored in 70% ethanol. Tissues were dehydrated by treating in a graded series of eth- anol. Tissues were cleared in toluene, infiltrated and embedded in paraffin blocks, and sectioned at a thickness of 5-7 ^m using a rotary micro- Figure 1. Photomicrograph of a cross-section of a tome. Ribbons were attached to slides with al- lesion elicited by Oligacanthorhynchus tortuosa in the bumin and stained folio wing Masson's trichrome intestine of a Virginia opossum (Didelphis virginiand) showing proboscis (arrow), necrotic abscess (asterisk), technique as described by Luna (1968). Two per- ring of collagen (C), region of active fibrocyte prolif- cent light green was substituted in place of aniline eration (arrowhead), muscularis (M), normal submu- blue to enhance staining of connective tissue. cosa (Sm), serosal side (S), and luminal side (L). Scale Stained sections were mounted in Canada bal- bar = 200 pm. Figure 1 appeared in Foundations of Parasitology, 5th edition, Wm. C. Brown, Publishers, sam and examined using light microscopy. and is used here with permission of the company. Worms were restricted to the anterior ¥2 of the small intestine, with most occurring in the an- terior '/3. Grossly, hard, white nodules were ap- mately 1.14 mm in diameter, were surrounded parent on the serosal surface of the intestine cor- by regions of dense connective tissue (collagen), responding to points of worm attachment. Pro- approximately 142 ^m wide. These regions of boscides were firmly embedded in the intestinal dense connective tissue, which appeared to have wall resulting in nodule formation; however, there effectively contained the abscesses, were inter- was no apparent evidence of hemorrhage as ob- spersed with small numbers of fibrocytes and served grossly by Babero (1957). At the base, fibroblasts. Spaces were noted between strands nodules ranged from approximately 1 to 5 mm of collagen, many of which appeared to be lym- in diameter. phatics. Bands of dense connective tissue were Lesions involved the mucosa, submucosa, and surrounded by regions of active fibroblast and muscularis; however, the serosa was intact and fibrocyte proliferation, approximately 169 ^m apparently unaffected (Fig. 1). Proboscides were wide, in which evidence of collagen synthesis contained within abscesses characterized by ne- could be observed. Within these regions, occa- crotic debris interspersed with many pycnotic sional plasma cells, lymphocytes, and mast cells nuclei, particularly abundant around the periph- were observed (approximately 1/0.133 mm2 [x 40 ery of abscesses. Small aggregations of collagen field]). These areas were infiltrated with many were present. No evidence of recent hemorrhage blood vessels and lymphatics. Lumina of arte- was observed. Abscesses, which were approxi- rioles were occluded by contraction of smooth

Copyright © 2011, The Helminthological Society of Washington RESEARCH NOTES 255 muscle in the arteriole wall. Abscesses along with ment of the worm does not occur after it has bands of connective tissue resulted in drastic en- established and embedded its proboscis into the largement of the submucosa to over 7 times its intestinal wall, unlike Moniliformis moniliformis normal width, resulting in formation of the gross- in rats, which was found to attach superficially, ly observable nodule. Width of the true sub- penetrating only the mucosa and tunica propria, mucosal region of unaffected tissue was approx- with no fibrosis (Taraschewski et al., 1989). Tar- imately 240 Aim, whereas that of affected regions aschewski et al. (1989) interpreted these lesions was 1.87 mm. In the regions of the lesions, both as evidence that worms frequently changed their longitudinal and smooth muscle layers of the sites of attachment. muscularis had been completely eroded; how- Pathologic changes induced by O. tortuosa in ever, the serosa remained intact and appeared to the opossum were similar, with notable excep- be unaffected. No evidence of hypertrophy of any tions, to those elicited by M. hirudinaceus and of the muscular layers in the vicinity of the le- M. ingens, which also belong to the family Oli- sions was observed. The mucosa and muscularis gacanthorhynchidae, in swine and , re- mucosae appeared to be intact on lumenal sides spectively (Nelson and Nickol, 1986). Nelson and of the lesions except for entry points of worms, Nickol (1986) reported extensive eosinophil pro- with thinning of the muscularis mucosae as an liferation and hypertrophy of the muscularis in apparent result of stretching. raccoons infected with M. ingens and congre- Histological series from which these data were gations of tissue macrophages, monocytes, and obtained were deposited in the Harold W. Man- plasma cells in swine infected with M. ingens. ter Laboratory, University of Nebraska, Lincoln, Such a pronounced cellular response was not as- Nebraska, and given accession Nos. HWML sociated with O. tortuosa infections in the opos- 37832-37834. sum. Additionally, hypertrophy of the muscu- Absence of polymorphonuclear leukocytes was laris was not observed. Instead, the muscularis indicative of chronic lesions. Observable pa- was virtually completely destroyed and had been thology may be solely accounted for by mechan- replaced by connective tissue. Both of these dif- ical damage resulting in subsequent fibrous nod- ferences may possibly be accounted for by du- ule formation. These findings corroborate the ration of infection. The observations of Nelson synopsis given by Nicholas (1967), who sum- and Nickol (1986) were based on lesions from a marized typical pathology of acanthocephalan raccoon infected with M. ingens, which was killed infection as traumatic injury as a result of the 63 days postinfection, and swine killed 3, 7, and proboscis penetrating deeply into the gut wall 14 days after experimental infection with M. in- leading to an inflammatory response with cel- gens. The duration of infection was not given for lular infiltration and the eventual formation of M. hirudinaceus in swine. It is possible that the a dense fibrous nodule around the proboscis. Se- reduced cellular response and pronounced ero- vere pathological manifestations associated with sion of the muscularis with subsequent fibrosis acanthocephalan infections often appear to be a in the present study, which are characteristic of result of caused by perforation of the chronic inflammation (McCutcheon, 1948), was serosa by the proboscis (Stunkard, 1965; Schmidt, a result of increased duration of infection. The 1972). Apparently this phenomenon was ob- lack of hemorrhage, as described by Babero served by Leidy (1850) for O. tortuosa. This is (1957), is further evidence of the chronicity of likely considering the extent of mechanical dam- the lesions. age found in the present study, including com- Pronounced absence of polymorphonuclear plete destruction of the muscularis. leukocytes suggests lack of a specific immune Lesions examined in this study were similar response to the presence of O. tortuosa with the to those described by Nelson and Nickol (1986) resultant inflammatory response and subsequent from domestic swine experimentally infected with fibrosis appearing to adequately contain the in- hirudinaceus, which also fection. Absence of polymorph proliferation sug- had nodule formation as a result of an increase gests a low degree of pathogenicity elicited by in size of the submucosa. Nelson and Nickol this parasite. One opossum examined was in- (1986) interpreted the preponderance of mono- fected with 99 O. tortuosa, mostly adults, and cytes and lymphocytes, fibrosis, and neovascu- exhibited no overt signs of illness. Histopatho- larization as evidence of a chronic lesion. logic changes elicited by O. tortuosa may be gen- Extensive fibrosis suggests that frequent move- eralized as a chronic inflammatory response to

Copyright © 2011, The Helminthological Society of Washington 256 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY OF WASHINGTON, 62(2), JUL 1995 mechanical trauma resulting from injury caused in W. A. D. Anderson, ed. Pathology. C. V. Mosby, by the proboscis, with subsequent fibrosis and St. Louis. Nelson, M. J., and B. B. Nickol. 1986. Survival of nodule formation, not unlike the inflammatory Macracanthorhynchus ingens in swine and histo- response elicited by an inanimate irritating body pathology of infection in swine and raccoons. as described by McCutcheon (1948). Journal of Parasitology 72:306-314. We thank Vernie L. Brown for aid in pro- Nicholas, W. L. 1967. The biology of the Acantho- curement of specimens, Scott Monks for tech- cephala. Advances in Parasitology 5:205-246. Richardson, D. J. 1993. of the Vir- nical assistance in slide preparation, William E. ginia opossum (Didelphis virginiana) in Arkansas, Moser, U.S. National Museum of Natural His- with a note on the life history of Centrorhynchus tory, Washington, D.C., for assistance in prep- wardae (Centrorhynchidae). Journal of the Hel- aration of this manuscript, and Dr. Amir N. minthological Society of Washington 60:128-130. , W. B. Owen, and D. E. Snyder. 1992. Hel- Hamir, School of Veterinary Medicine, Univer- minth parasites of the raccoon (Procyon lotor) from sity of Pennsylvania, for critical review of the north central Arkansas. Journal of Parasitology 7 8: manuscript. 163-166. Schmidt, G. D. 1972. Acanthocephala of captive pri- Literature Cited mates. Pages 144-156 in R. N. T-W-Fiennes, ed. Pathology of Simian Primates. Vol. 2. S. Karger, Babero, B. B. 1957. Some helminths from Illinois Basel. opossums. Journal of Parasitology 43:232. Stunkard, H. W. 1965. New intermediate hosts in . 1960. Further studies on helminths of the the life cycle of elegans (Diesing, opossum Didelphis virginiana, with a description 1851), an acanthocephalan parasite of primates. of a new species from the . Journal of Para- Journal of Parasitology 51:645-649. sitology 46:455-463. Taraschewski, H., C. Sagani, and H. Mehlhorn. 1989. Feldman, D. B., J. A. Moore, M. W. Harris, and J. L. Ultrastructural study of the host-parasite interface Self. 1972. Characteristics of common hel- of Moniliformis moniliformis (Archiacanthocc- minths of the Virginia opossum (Didelphis virgi- phala) in laboratory-infected rats. Journal of Par- niana) from North Carolina. Laboratory asitology 75:288-296. Science 22:183-189. Van Cleave, H. J. 1924. A critical study of the Acan- Leidy, J. 1850. Contributions to helminthology. Pro- thocephala described and identified by Joseph Lei- ceedings of the Academy of Natural Sciences of dy. Proceedings of the Academy of Natural Sci- Philadelphia 5:96-98. ences of Philadelphia 76:279-334. Luna, L. G. 1968. Manual of Histologic Staining . 1953. Acanthocephala of North American Methods of the Armed Forces Institute of Pa- mammals. Illinois Biological Monographs 23:1- thology, 3rd ed. McGraw Hill, New York. 258 pp. 179. McCutcheon, M. 1948. Inflammation. Pages 14-66

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