EXPERIMENTAL
PARASITOLOGY
Anisakis RICHARD Department Alabama
simplex:
70, 305-313 (1990)
Histopathological Changes Infected CBNJ Mice
E. JONES, THOMAS
of Structural 36688, U.S.A.,
L. DEARDORFF,*”
and Cellular Biology, University and *Fishery Research Branch, Dauphin Island, Alabama
in Experimentally
AND STEPHEN
G. KAYES
of South Alabama College of Medicine, Mobile, U.S. Food and Drug Administration, Box 158, 36528, U.S.A.
JONES,R. E.,DEARDORFF,T. L., AND KAYES, S. G. 1990. Anisakis simplex: Histopathological changes in experimentally infected CBA/J mice. Experimental Parasitology 70, 305-313. Third-stage juveniles (L,) of Anisakis simplex, surgically implanted into the abdominal cavity of CBA/J mice and necropsied at 7, 14, or 21 days postinfection (PI), embedded in the gut mesentery and only rarely invaded viscera. Histologically, intense aggregations of neutrophils adjacent to the parasites were noted at Day 7 PI. At Day 14 PI, mature granulomata consisting mostly of eosinophils and large numbers of tibroblasts and associated collagen were observed. Granulocytes and occasionally multinucleate giant cells occupied the host-parasite interface. At 21 day PI, lesions displayed the predominance of connective tissue. Multinucleate giant cells were found adjacent to the L, with eosinophils adjacent to parasite remnants or scattered within the walls of the granulomata. Most L3 were viable at Days 7 and 14 PI; however, at Day 21 PI the L, were dead and invaded by inflammatory cells. Hematological findings indicated that infected mice had a neutrophilia of varying magnitude regardless of the number of worms implanted. Eosinophil levels as a percentage of the total leukocyte pool in peripheral blood always remained at or below normal limits. On Days 7 and 14 PI, the peripheral blood showed an increase in neutrophils that began to return to normal values at 21 day PI. Conversely, peripheral blood eosinophils decreased on Days 7 and 14 PI and returned to normal values on Day 21 PI. Surgical implantation of A. simplex L, into mice produced both a hematological and histological picture consistent with that seen in human anisakiasis. o 1990 Academic press, hc. INDEX DESCRIPTORS AND ABBREVIATIONS: Nematode; Parasite: Anisakis simplex; Histopathological changes; Eosinophil; CBA/J mice; Experimental anisakiasis; Marine fishes; Public health; Polymorphonuclear neutrophil (PMN); Gastrointestinal ((31); Third-stage juvenile (L,); Excretory-secretory (ES); Phosphate-buffered saline (PBS); Postinfection (PI).
The third-stage juvenile (L3) of the marine ascaridoid Anisakis simplex may represent a significant health threat to individuals who ingest raw or undercooked seafood products that have been previously infected. In human anisakiasis, the L, are capable of penetrating the tissues of the GI tract (Bier et al. 1987; Deardorff and Overstreet 1990; Oshima 1972). Although the L, have been recovered from the small and large intestine, pancreas, mesentery, and other tissues (Yokogawa and Yoshimura i To whom requests for reprints dressed.
should be ad-
1967), they are most often found within the mucosa and submucosa of the GI tract. Because of variation in the sites of invasion and host response, anisakiasis mimics a wide variety of clinical manifestations (e.g., Crohn’s disease, gastric tumors and cancer, ileitis, tuberculous peritonitis, acute appendicitis, rectal carcinoma) that confuse the diagnosis. For example, one hospital in Japan reported 27 preoperative clinical diagnoses for 219 cases that were postoperatively confirmed to be gastric or intestinal anisakiasis (Ishikura 1969). Misdiagnoses have also occurred in the U.S. (Sakanari et al. 1988). To increase our understanding of the im305 0014-4894/90 $3.00 Copytiht All rights
8 1990 by Academic Press, Inc. of reproduction in any form reserved.
306
JONES,DEARDORFF,
mune responses and immunopathological changes associated with this zoonotic infection and develop a serodiagnostic assay specific for the L, of A. simplex, we are attempting to develop an analog of the human infection. Previous infectivity studies have used juveniles belonging to several anisakid genera and gavaged the worms into the stomach of guinea pigs, rats, rabbits, minks, dog, and monkeys (e.g., Myers 1963; Asami and Inoshita 1967; Deardorff et al. 1983; Overstreet and Meyer 1981; Gibson 1970; Ruitenberg 1970; Croll et al. 1980; Margolis and Beverley-Burton 1977). The emphasis of these studies was to document the invasive potential of the helminths. Fewer studies have been concerned with documenting the immune responses and inflammatory processes in the infected animals (e.g., Ruitenberg 1970; Suzuki 1968; Suzuki et al. 1969; 1971; Bier and Raybourne 1988). Although much can be learned from these studies, the use of outbred strains of animals has produced conflicting and confusing results. Because of the size of the mice in relation to the large L,, they have not been considered as a model for infection. However, mice offer a significant advantage over the other experimental animals because inbred strains are readily available and allow for a more accurate and controlled model to study immunobiologic responses. Studies by Kayes (1984) and Kayes et al. (1986, 1987) with another pathogenic ascaridoid nematode, Tuxocuru cunis, suggested that the inbred CBA/J mouse strain may represent a good analog for human anisakiasis. As part of our studies to evaluate the CBA/J mouse as a model for the human infection, we report herein our findings on the histopathological alterations seen in experimental murine anisakiasis. MATERIALSANDMETHODS Parasite. L3 of A. simplex were removed from the viscera of Pacific rockfishes (Sebasres spp.) or Pacific salmon (Oncorhynchus spp.) collected from the Pacific
AND KAYES Northwest in 1988. L, were isolated from fish viscera by the pepsin-hydrochloric acid process (Stem et al. 1958) as modified by Deardorff and Throm (1988), rinsed several times in sterile PBS, and maintained at ambient temperature in RPM1 1640 tissue culture medium (Sigma Chemical Co., St. Louis, MO) containing 50 &ml gentamicin sulfate (Sigma Chemical Co.). Mice. Female CBA/J mice, obtained from the Jackson Laboratories (Bar Harbor, ME), were maintained on standard rodent chow and water ad libitum in the Animal Health and Resources facility of the College of Medicine, University of South Alabama. These facilities meet the guidelines established by the National Institutes of Health in the “Guide for the Care and Use of Laboratory Animals.” All mice were obtained as weanlings (weight, 12-15 g) and allowed a minimum of 7 days to acclimate to these facilities prior to inclusion into the experiments. Surgical implantation and procedures. Within 24 hr after isolation of the L, from the fish tissues, surgical implantation was performed. L, were rinsed in sterile RPM1 1640 without antibiotics prior to placement into the abdominal cavity of the mice. Mice were anesthetized with ether vapor, a surgical incision was made over the lateral peritoneal cavity, 2,5, or 10 viable and undamaged L, were placed in the abdominal cavity, and the wound was closed with 9-mm stainless-steel wound clips. A total of 27 mice were used; three mice for each infection size and time period. Additionally, three control animals underwent sham surgery. Mice were necropsied at Days 7, 14, or 21 PI. Peripheral blood smears were made, stained with WrightGiemsa stain, and the percentage of each leukocyte type was determined. Gross examinations of the peritoneal cavity were performed to recover tissues or organs displaying obvious signs of gross pathological change or attached worms and the tissues were fixed in 10% phosphate-buffered formalin. Worms removed for the peritoneal cavity were considered viable if movement was observed prior to fixation or by microscopic evaluation of sections of parasites. Sections that revealed uncompromised structural integrity were regarded as viable. All necropsy material was processed and stained using standard procedures. RESULTS
Mice appeared to tolerate the implanted juveniles quite well. No abnormal behavior or change in food or water intake were observed during the experimental period in mice infected with L3 of A. simplex. Upon necropsy at each time period, L, were principally found embedded in the mesentery. Rarely were L3 found in viscera. The majority of worms recovered at Days 7 and 14
EXPERIMENTAL
MURINE
PI \ were viable. LJ, recovered at Day 21 PI, we1‘e dead and invaded by inflammatory cell s. Ait Day 7 PI, lesions exhibited an intense,
ANISAKIASIS
acute inflammatory reaction, princip; composed of neutrophils, adjacent to parasite (Fig. I). Chronic inflammatory ements represented by fibroblasts and
FIGS. l-3. Murine anisakiasis at Day 7 PI. (1) Normal mesenteric architecture has been obliterated by an intense, acute inflammatory process surrounding tangential section of larval worm (X 100). (2) Higher magnification of area delineated by box in Fig. 1 showing aggregations of neutrophils adjacent to the parasite. Arrow indicates thin rim of fibroblasts along with secreted nascent collagen (x250). (3) Section of pancreas collateral to parasite-associated lesion with wedge-shaped eosinophilic infiltrate (X 100).
307 ally the elas-
308
JONES,
DEARDORFF,
sociated collagen (Fig. 2) were seen surrounding the acute reaction. Perivascular cuffing of eosinophils around capillaries and venules adjacent to the inflammatory nidus was observed. Eosinophil recruitment was not limited to microvasculature peripheral to the inflammatory milieu surrounding the parasite. For example, sections taken through a pancreas removed serendipidously along with a parasite-associated lesion revealed a wedgeshaped eosinophilic infiltrate into this organ, even though the pancreas had not been invaded by the parasite (Fig. 3). Associated mesenteric architecture was intact and no evidence of previous disruption and subsequent healing, such as accumulations of neutrophils and fibroblasts, were observed among the adipocytes, except in immediate proximity of the worms. Tissue damage, therefore, could be found in collateral areas where no worms were located. At Day 14 PI, lesions were characterized by well-formed, fibrotically encapsulated granulomas (Fig. 4). The fibrotic wall of the granuloma contained robust profiles of libroblasts consistent with the deposition of large quantities of nascent collagens. The majority of inflammatory cells adjacent to the worms were eosinophils; the remaining cells were fibroblasts or multinucleate giant cells (Fig. 5). Some of the parasites contained within Day 14 lesions were dead. Large granulomas persisted around worm remnants at day 21 PI (Fig. 6). These granulomata were composed of a thick fibrous wall with multinucleate giant cells of both foreign body and Langhans type comprising the inner surface of the granulomaparasite interface (Figs. 7 and 8). Eosinophils were present in varying numbers within these lesions but were most abundant when associated with parasite remnants . The relationship of PMNs and eosinophils in the peripheral blood of infected mice during the course of the experiment is shown in Fig. 9. Peripheral blood PMNs
AND
KAYES
peaked at Day 14 PI and comprised almost 50% of the total circulating white cells. Maximum neutrophilia in mice infected with 10 worms at Day 14 PI was twofold that of control mice. At Day 21 PI, the PMNs began to drop to normal values. Conversely, eosinophils decreased sixfold on Day 7 PI but returned to normal levels (1.6% of total WBC) by Day 21 PI. Mice receiving sham operations consistently maintained total leukocyte counts and differential profiles within normal limits (data not shown). DISCUSSION
The histologic changes seen in the surgically infected CBA/J mice closely resembled those reported in both orally infected laboratory animals and humans. Eosinophilic infiltration, fibroblast proliferation, and granuloma formation are salient features in all infections. The lesions caused by juvenile A. simplex have been diagnostically confused with and clinically associated with malignant cells. Of the 153 cases of gastric anisakiasis reported by Ishikura (1969) that occurred in patients at one Japanese hospital, for example, 63 (41%) were preoperatively diagnosed as gastric cancer or tumor. Hayakawa et al. (1970) and Tsutsumi and Fujimoto (1983) have each reported a human case involving the presence of cancer cells (type Ha) adjacent to the invading L3. Tsutsumi and Fujimoto (1983) concluded that the L, probably did not induce the early cancer; rather, the cancer presented an entry site for the L,. The association of nematodes with carcinomas, however, is not new (Bonne and Sandground 1939; Smetana and Orihel 1969). The finding that sections of pancreas contained eosinophilic infiltrates in the absence of L, suggests that the excretorysecretory (ES) antigens may be active in eosinophil recruitment to remote sites (Tanaka and Torisu 1982). Further, the nematode ES antigens may be involved
EXPERIMENTAL
MURINE
ANISAKIASIS
FIGS. 4 AND 5. Murine anisakiasis at Day 14 PI. (4) A well-formed fibrotic granuloma with outer wall of granuloma showing exuberant fibroblast proliferation (x250). (5) Higher magnification of Fig. 4 showing eosinophils (cells with annular nuclei) as predominant granulocyte cell type (x 1000).
309
310
JONES,
DEARDORFF,
AND
KAYES
FIGS. f&8. Murine anisakiasis at Day 21 PI. (6) Extensive bands of fibroblasts and collagen in granulomas formed around parasitic debris. Intense accumulations of eosinophils remain in proximity to parasite remnants (X 100). (7) Individual granuloma composed of thick fibrotic wall surrounding larva (x250). (8) Higher magnification of Fig. 7 showing granuloma-parasite interface. Note that multinucleate giant cells comprise the inner surface of the granuloma (X 1000).
EXPERIMENTAL 5%
MURINE
311
ANISAKIASIS
r
150%
,
0% ’ DAY
I 0
DAY
I 7
DAY
I DAY
14
’ 0% 21
FIG. 9. Changes in the mean peripheral blood eosinophil (left scale; n ) and neutrophil (right scale; 0) counts, expressed as percentage of total leukocytes, at Day 21 PI in mice that received 10 L,. Each point is the mean of three mice and vertical bars represent SE of the mean.
with the exuberant tibroblast proliferation and secretion that we observed juxtaposed to the encapsulated L,. Juvenile A. simplex ES antigens have potent bioactive properties (Raybourne et al. 1983, 1986) and have been demonstrated to be released in the tissues by the invading L3 (Sakanari et al. 1988). The possibility that these ES antigens possess a fibroblast growth factor is being investigated. Fibroblast proliferation may be a host-induced protective reaction that contributes to the isolation of the penetrating L, and associated ES antigens. Recent findings indicate protease activity associated with these parasite products (Matthews 1982; Sakanari and McKerrow 1988). Peripheral blood eosinophilia has been reported to range from 4 to 41% in human anisakiasis (i.e., Sakanari el al. 1988) but was not evident in our experimental mice. We attribute this phenomena to our method of infection. Per 0s infections allow for penetration into or through the GI tract which would rapidly stimulate various immunobiologic responses of the host leading to enhanced eosinophilopoiesis. Induction of an eosinophilic response, for example, in in-
fections
with the juveniles of Trichinella the parasites to become embolized in the lungs of the host (Basten et al. 1970). Without pulmonary embolization, which may be precluded by intramuscular injection of T. spiralis juveniles rather than per OS, no eosinophilia occurs. With the surgical method of infection employed in our study, the L, showed no proclivity for the invasion of viscera. We conclude the histopathologic events in human anisakiasis and our experimental model are similar. Lesions with eosinophilic components and exuberant fibroblast proliferation and collagen secretion are common in both hosts; however, in the mouse it occurs in the absence of a peripheral blood eosinophilia. spiralis requires
ACKNOWLEDGMENTS We gratefully acknowledge Ms. Martha Stober (U.S.A., Mobile) for technical assistance. Fishes were collected by FDA inspector Mr. Dick Throm (Seattle District) with the cooperation of Mr. Mike Kinkade and the stalf at Bomstein Seafood Inc., Bellingham, Washington, and Mr. John W. McCallum and the staff at The Pacific Salmon Co., Inc., Seattle, Washington, This study was supported in part by NIH Grant AI
JONES, DEARDORFF,
312
19968 and American Heart Association, Alabama Affiliate, Inc., Grant 870834 awarded to S.G.K. REFERENCES ASAMI, K., AND INOSHITA, Y. 1%7. Experimental anisakiasis in guinea pigs: Factors influencing infection of larvae in the host. Japanese Journal of Parasitology 16, 415-422. BASTEN, A. B., BOYER, M. H., AND BEESON, P. B. 1970. Mechanism of eosinophilia. I. Factors affecting the eosinophil response of rats to Trichinella spiralis. Journal of Experimental Medicine 131, 12711287. BIER, J. W., DEARDORFF, T. L., JACKSON,G. J., AND RAYBOURNE, R. B. 1987. Human anisakiasis. In “Bailliere’s Clinical Tropical Medicine and Communicable Diseases: Intestinal Helminthic Infections” (Z. S. Pawlowski, Ed.), Vol. 2, pp. 723-733. Saunders, Philadelphia. BIER, J. W., AND RAYBOURNE, R. B. 1988. Anisakis simplex. (Nematoda: Ascaridoidea): Formation of immunogenic attachment caps in pigs. Proceedings of the Helminthological
Society
of Washington
55,
91-94. BONNE, C., AND SANDGROUND, J. H. 1939. On the production of gastric tumors, bordering on malignancy in Javanese monkeys through the agency of Nochtia nochti, a parasitic nematode. American Journal of Cancer 37, 173-185. CROLL, N. A., MA, K., SMITH, J. M., AND SUKHDEO, M. V. K. 1980. Phocanema decipiens: Intestinal penetration in the laboratory rat. Experimental Parasitology 50, 145-154. DEARDORFF, T. L., KLIKS, M. M., AND DESOWITZ, R. S. 1983. Histopathology induced by larval Terranova (type HA) (Nematoda: Anisakinae) in experimentally infected rats. Journal of Parasitology 69, 191-195. DEARDORFF, T. L., AND OVERSTREET, R. M. 1990. Seafood-transmitted zoonoses in the United States: The fishes, the dishes, and the worms. In “Microbiology of Marine Food Products (C. R. Hackney and D. R. Ward, Eds.), Van Nostrand/Reinhold, New York. DEARDORFF, T. L., AND THROM, R. 1988. Commercial blast-freezing of third-stage Anisakis simplex larvae encapsulated in salmon and rocklish. Journal of Parasitology
74, 600-603.
GIBSON, D. I. 1970. Aspects of the development of “herringworm” (Anisakis sp. larva) in experimentally infected rats. Nytt Magasin for Zoologi 18, 175-87. HAYAKAWA, M., SUZUKI, K., MAEDA, T., OIKAWA, K., AND ISHIDATE, T. 1970. A case of Ha type early gastric cancer associated with eosinophilic granuloma. Stomach and Intestine 5, 223-227.
AND KAYES
ISHIKURA, H. 1969. Occurrence of anisakiasis and its clinical presentation. Saishin Igaku 24, 357-365. KAYES, S. G. 1984. Spleen cell responses in experimental murine toxocariasis. Journal of Parasitology 70, 522-529. KAYES, S. G., JONES, R. E., AND OMHOLT, P. E. 1987. Use of bronchoalveolar lavage to compare local pulmonary immunity with the systemic immune response of Toxocara canis-infected mice. Infection and immunity 55, 2132-2136. KAYES, S. G., JONES, R. E., AND OMHOLT, P. E. 1986. Pulmonary granuloma formation in murine toxocariasis: Transfer of granulomatous hypersensitivity using bronchoalveolar lavage cells. Journal of Parasitology
74, 950-956.
MARGOLIS, L., AND BEVERLEY-BURTON, M. 1977. Response of mink (Mustela vison) to larval Anisakis simplex (Nematoda: Ascaridida). International Journal
for
Parasitology
I, 269-273.
MATTHEWS, B. E. 1982. Behavior and enzyme release by Anisakis sp. larvae (Nematoda: Ascaridida). Journal of Helminthology 56, 177-183. MYERS, B. J. 1%3. The migration ofdnisakis-type larvae in experimental animals. Canadian Journal of Zoology 41, 147-148. OSHIMA, T. 1972. Anisakis and anisakiasis in Japan and adjacent area. In “Progress of Medical Parasitology in Japan,” Vol. 4, pp. 301-393. Meguro Parasitological Museum, Tokyo, Japan. OVERSTREET, R. M., AND MEYER, G. W. 1981. Hemorrhagic lesions in stomach of rhesus monkey caused by a piscine ascaridoid nematode. Journal of Parasitology
67, 226-235.
RAYBOURNE, R. B., DEARDORFF, T. L., AND BIER, J. W. 1986. Anisakis simplex: Larval excretorysecretory protein production and cytostatic action in mammalian cell cultures. Experimental Parasitology 62, 92-97. RAYBOURNE, R. B., DESOWITZ, R. S., KLIKS, M. M., AND DEARDORFF, T. L. 1983. Anisakis simplex and Terranova sp.: Inhibition by larval excretorysecretory products of mitogen-induced rodent lymphoblast proliferation. Experimental Parasitology 55, 289-298. RUITENBERG, E. J. 1970. Anisakiasis: Pathogenesis, Serodiagnosis and Prevention, 138 p. Thesis, Rijksuniversiteit, Utrecht. SAKANARI, J. A., LOINAZ, H. M., DEARDORFF, T. L., RAYBOURNE, R. B., MCKERROW, J. H., AND FRIERSON,J. G. 1988. Intestinal anisakiasis: A case diagnosed by morphologic and immunologic methods. American Journal of Clinical Pathology 90, 107-113. SAKANARI, J. A., AND MCKERROW, J. H. 1988. Characterization of the secreted proteases of Anisakis sp. In “Proceedings, 63rd Annual Meeting of The
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MURINE
American Society of Parasitologists, WinstonSalem, North Carolina,” p. 43. SMETANA, H. F., AND ORIEHEL, T. C. 1%9. Gastric papillomata in Macaca speciosa induced by Nochtia nochti (Nematoda: Trichostrongyloidea). Journal of Parasitology 55, 349-35 1. STERN, J. A., CHAKRAVARTI, D., UZMANN, J. R., AND HESSELHOLT, N. M. 1958. “Rapid Counting of Nematoda in Salmon by Peptic Digestion,” pp. 1-S. U.S. Fish and Wildlife Service, Special Scientific Report-Fisheries No. 255. SUZUKI, T. 1%8. Studies on the immunological diagnosis of anisakiasis. I. Antigenic analysis of Anisakis larvae by means of electrophoresis. Japanese Journal of Parasitology 17, 213-220. SUZUKI, T., SHIRAKI, T., AND OTSURU, M. 1969. Studies on the immunological diagnosis of anisakiasis. II. Isolation and purification of Anisakis antigen. Japanese Journal of Parasitology 18, 232-240. SUZUKI, T., SHIRAKI, T., AND OTSURU, M. 1971. Im-
313
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munodiagnosis of anisakiasis, with special reference to purification of specific antigen. Chinese Journal of Microbiology 4, 217-231. TANAKA, J., AND TORISU, M. 1982. Anisakis and eosinophils. I. Detection of a soluble factor selectively chemotactic for eosinophils in the extract from Anisakis larvae. Journal of Immunology 120, 745-749.
TSUTSUMI, Y., AND FUIIMOTO, Y. 1983. Early gastric cancer superimposed on infestation of an Anisakislike larva: A case report. Tokai Journal of Experimental
and Clinical
Medicine
8, 265-273.
YOKOGAWA, M., AND YOSHIMURA, H. 1967. Clinicopathologic studies on larval anisakiasis in Japan. American Journal giene 16, 723-728.
of Tropical
Medicine
and
Hy-
Received 5 July 1989; accepted with revision 30 October 1989
PARASITOLOGY
Anisakis RICHARD Department Alabama
simplex:
70, 305-313 (1990)
Histopathological Changes Infected CBNJ Mice
E. JONES, THOMAS
of Structural 36688, U.S.A.,
L. DEARDORFF,*”
and Cellular Biology, University and *Fishery Research Branch, Dauphin Island, Alabama
in Experimentally
AND STEPHEN
G. KAYES
of South Alabama College of Medicine, Mobile, U.S. Food and Drug Administration, Box 158, 36528, U.S.A.
JONES,R. E.,DEARDORFF,T. L., AND KAYES, S. G. 1990. Anisakis simplex: Histopathological changes in experimentally infected CBA/J mice. Experimental Parasitology 70, 305-313. Third-stage juveniles (L,) of Anisakis simplex, surgically implanted into the abdominal cavity of CBA/J mice and necropsied at 7, 14, or 21 days postinfection (PI), embedded in the gut mesentery and only rarely invaded viscera. Histologically, intense aggregations of neutrophils adjacent to the parasites were noted at Day 7 PI. At Day 14 PI, mature granulomata consisting mostly of eosinophils and large numbers of tibroblasts and associated collagen were observed. Granulocytes and occasionally multinucleate giant cells occupied the host-parasite interface. At 21 day PI, lesions displayed the predominance of connective tissue. Multinucleate giant cells were found adjacent to the L, with eosinophils adjacent to parasite remnants or scattered within the walls of the granulomata. Most L3 were viable at Days 7 and 14 PI; however, at Day 21 PI the L, were dead and invaded by inflammatory cells. Hematological findings indicated that infected mice had a neutrophilia of varying magnitude regardless of the number of worms implanted. Eosinophil levels as a percentage of the total leukocyte pool in peripheral blood always remained at or below normal limits. On Days 7 and 14 PI, the peripheral blood showed an increase in neutrophils that began to return to normal values at 21 day PI. Conversely, peripheral blood eosinophils decreased on Days 7 and 14 PI and returned to normal values on Day 21 PI. Surgical implantation of A. simplex L, into mice produced both a hematological and histological picture consistent with that seen in human anisakiasis. o 1990 Academic press, hc. INDEX DESCRIPTORS AND ABBREVIATIONS: Nematode; Parasite: Anisakis simplex; Histopathological changes; Eosinophil; CBA/J mice; Experimental anisakiasis; Marine fishes; Public health; Polymorphonuclear neutrophil (PMN); Gastrointestinal ((31); Third-stage juvenile (L,); Excretory-secretory (ES); Phosphate-buffered saline (PBS); Postinfection (PI).
The third-stage juvenile (L3) of the marine ascaridoid Anisakis simplex may represent a significant health threat to individuals who ingest raw or undercooked seafood products that have been previously infected. In human anisakiasis, the L, are capable of penetrating the tissues of the GI tract (Bier et al. 1987; Deardorff and Overstreet 1990; Oshima 1972). Although the L, have been recovered from the small and large intestine, pancreas, mesentery, and other tissues (Yokogawa and Yoshimura i To whom requests for reprints dressed.
should be ad-
1967), they are most often found within the mucosa and submucosa of the GI tract. Because of variation in the sites of invasion and host response, anisakiasis mimics a wide variety of clinical manifestations (e.g., Crohn’s disease, gastric tumors and cancer, ileitis, tuberculous peritonitis, acute appendicitis, rectal carcinoma) that confuse the diagnosis. For example, one hospital in Japan reported 27 preoperative clinical diagnoses for 219 cases that were postoperatively confirmed to be gastric or intestinal anisakiasis (Ishikura 1969). Misdiagnoses have also occurred in the U.S. (Sakanari et al. 1988). To increase our understanding of the im305 0014-4894/90 $3.00 Copytiht All rights
8 1990 by Academic Press, Inc. of reproduction in any form reserved.
306
JONES,DEARDORFF,
mune responses and immunopathological changes associated with this zoonotic infection and develop a serodiagnostic assay specific for the L, of A. simplex, we are attempting to develop an analog of the human infection. Previous infectivity studies have used juveniles belonging to several anisakid genera and gavaged the worms into the stomach of guinea pigs, rats, rabbits, minks, dog, and monkeys (e.g., Myers 1963; Asami and Inoshita 1967; Deardorff et al. 1983; Overstreet and Meyer 1981; Gibson 1970; Ruitenberg 1970; Croll et al. 1980; Margolis and Beverley-Burton 1977). The emphasis of these studies was to document the invasive potential of the helminths. Fewer studies have been concerned with documenting the immune responses and inflammatory processes in the infected animals (e.g., Ruitenberg 1970; Suzuki 1968; Suzuki et al. 1969; 1971; Bier and Raybourne 1988). Although much can be learned from these studies, the use of outbred strains of animals has produced conflicting and confusing results. Because of the size of the mice in relation to the large L,, they have not been considered as a model for infection. However, mice offer a significant advantage over the other experimental animals because inbred strains are readily available and allow for a more accurate and controlled model to study immunobiologic responses. Studies by Kayes (1984) and Kayes et al. (1986, 1987) with another pathogenic ascaridoid nematode, Tuxocuru cunis, suggested that the inbred CBA/J mouse strain may represent a good analog for human anisakiasis. As part of our studies to evaluate the CBA/J mouse as a model for the human infection, we report herein our findings on the histopathological alterations seen in experimental murine anisakiasis. MATERIALSANDMETHODS Parasite. L3 of A. simplex were removed from the viscera of Pacific rockfishes (Sebasres spp.) or Pacific salmon (Oncorhynchus spp.) collected from the Pacific
AND KAYES Northwest in 1988. L, were isolated from fish viscera by the pepsin-hydrochloric acid process (Stem et al. 1958) as modified by Deardorff and Throm (1988), rinsed several times in sterile PBS, and maintained at ambient temperature in RPM1 1640 tissue culture medium (Sigma Chemical Co., St. Louis, MO) containing 50 &ml gentamicin sulfate (Sigma Chemical Co.). Mice. Female CBA/J mice, obtained from the Jackson Laboratories (Bar Harbor, ME), were maintained on standard rodent chow and water ad libitum in the Animal Health and Resources facility of the College of Medicine, University of South Alabama. These facilities meet the guidelines established by the National Institutes of Health in the “Guide for the Care and Use of Laboratory Animals.” All mice were obtained as weanlings (weight, 12-15 g) and allowed a minimum of 7 days to acclimate to these facilities prior to inclusion into the experiments. Surgical implantation and procedures. Within 24 hr after isolation of the L, from the fish tissues, surgical implantation was performed. L, were rinsed in sterile RPM1 1640 without antibiotics prior to placement into the abdominal cavity of the mice. Mice were anesthetized with ether vapor, a surgical incision was made over the lateral peritoneal cavity, 2,5, or 10 viable and undamaged L, were placed in the abdominal cavity, and the wound was closed with 9-mm stainless-steel wound clips. A total of 27 mice were used; three mice for each infection size and time period. Additionally, three control animals underwent sham surgery. Mice were necropsied at Days 7, 14, or 21 PI. Peripheral blood smears were made, stained with WrightGiemsa stain, and the percentage of each leukocyte type was determined. Gross examinations of the peritoneal cavity were performed to recover tissues or organs displaying obvious signs of gross pathological change or attached worms and the tissues were fixed in 10% phosphate-buffered formalin. Worms removed for the peritoneal cavity were considered viable if movement was observed prior to fixation or by microscopic evaluation of sections of parasites. Sections that revealed uncompromised structural integrity were regarded as viable. All necropsy material was processed and stained using standard procedures. RESULTS
Mice appeared to tolerate the implanted juveniles quite well. No abnormal behavior or change in food or water intake were observed during the experimental period in mice infected with L3 of A. simplex. Upon necropsy at each time period, L, were principally found embedded in the mesentery. Rarely were L3 found in viscera. The majority of worms recovered at Days 7 and 14
EXPERIMENTAL
MURINE
PI \ were viable. LJ, recovered at Day 21 PI, we1‘e dead and invaded by inflammatory cell s. Ait Day 7 PI, lesions exhibited an intense,
ANISAKIASIS
acute inflammatory reaction, princip; composed of neutrophils, adjacent to parasite (Fig. I). Chronic inflammatory ements represented by fibroblasts and
FIGS. l-3. Murine anisakiasis at Day 7 PI. (1) Normal mesenteric architecture has been obliterated by an intense, acute inflammatory process surrounding tangential section of larval worm (X 100). (2) Higher magnification of area delineated by box in Fig. 1 showing aggregations of neutrophils adjacent to the parasite. Arrow indicates thin rim of fibroblasts along with secreted nascent collagen (x250). (3) Section of pancreas collateral to parasite-associated lesion with wedge-shaped eosinophilic infiltrate (X 100).
307 ally the elas-
308
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DEARDORFF,
sociated collagen (Fig. 2) were seen surrounding the acute reaction. Perivascular cuffing of eosinophils around capillaries and venules adjacent to the inflammatory nidus was observed. Eosinophil recruitment was not limited to microvasculature peripheral to the inflammatory milieu surrounding the parasite. For example, sections taken through a pancreas removed serendipidously along with a parasite-associated lesion revealed a wedgeshaped eosinophilic infiltrate into this organ, even though the pancreas had not been invaded by the parasite (Fig. 3). Associated mesenteric architecture was intact and no evidence of previous disruption and subsequent healing, such as accumulations of neutrophils and fibroblasts, were observed among the adipocytes, except in immediate proximity of the worms. Tissue damage, therefore, could be found in collateral areas where no worms were located. At Day 14 PI, lesions were characterized by well-formed, fibrotically encapsulated granulomas (Fig. 4). The fibrotic wall of the granuloma contained robust profiles of libroblasts consistent with the deposition of large quantities of nascent collagens. The majority of inflammatory cells adjacent to the worms were eosinophils; the remaining cells were fibroblasts or multinucleate giant cells (Fig. 5). Some of the parasites contained within Day 14 lesions were dead. Large granulomas persisted around worm remnants at day 21 PI (Fig. 6). These granulomata were composed of a thick fibrous wall with multinucleate giant cells of both foreign body and Langhans type comprising the inner surface of the granulomaparasite interface (Figs. 7 and 8). Eosinophils were present in varying numbers within these lesions but were most abundant when associated with parasite remnants . The relationship of PMNs and eosinophils in the peripheral blood of infected mice during the course of the experiment is shown in Fig. 9. Peripheral blood PMNs
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peaked at Day 14 PI and comprised almost 50% of the total circulating white cells. Maximum neutrophilia in mice infected with 10 worms at Day 14 PI was twofold that of control mice. At Day 21 PI, the PMNs began to drop to normal values. Conversely, eosinophils decreased sixfold on Day 7 PI but returned to normal levels (1.6% of total WBC) by Day 21 PI. Mice receiving sham operations consistently maintained total leukocyte counts and differential profiles within normal limits (data not shown). DISCUSSION
The histologic changes seen in the surgically infected CBA/J mice closely resembled those reported in both orally infected laboratory animals and humans. Eosinophilic infiltration, fibroblast proliferation, and granuloma formation are salient features in all infections. The lesions caused by juvenile A. simplex have been diagnostically confused with and clinically associated with malignant cells. Of the 153 cases of gastric anisakiasis reported by Ishikura (1969) that occurred in patients at one Japanese hospital, for example, 63 (41%) were preoperatively diagnosed as gastric cancer or tumor. Hayakawa et al. (1970) and Tsutsumi and Fujimoto (1983) have each reported a human case involving the presence of cancer cells (type Ha) adjacent to the invading L3. Tsutsumi and Fujimoto (1983) concluded that the L, probably did not induce the early cancer; rather, the cancer presented an entry site for the L,. The association of nematodes with carcinomas, however, is not new (Bonne and Sandground 1939; Smetana and Orihel 1969). The finding that sections of pancreas contained eosinophilic infiltrates in the absence of L, suggests that the excretorysecretory (ES) antigens may be active in eosinophil recruitment to remote sites (Tanaka and Torisu 1982). Further, the nematode ES antigens may be involved
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FIGS. 4 AND 5. Murine anisakiasis at Day 14 PI. (4) A well-formed fibrotic granuloma with outer wall of granuloma showing exuberant fibroblast proliferation (x250). (5) Higher magnification of Fig. 4 showing eosinophils (cells with annular nuclei) as predominant granulocyte cell type (x 1000).
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FIGS. f&8. Murine anisakiasis at Day 21 PI. (6) Extensive bands of fibroblasts and collagen in granulomas formed around parasitic debris. Intense accumulations of eosinophils remain in proximity to parasite remnants (X 100). (7) Individual granuloma composed of thick fibrotic wall surrounding larva (x250). (8) Higher magnification of Fig. 7 showing granuloma-parasite interface. Note that multinucleate giant cells comprise the inner surface of the granuloma (X 1000).
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r
150%
,
0% ’ DAY
I 0
DAY
I 7
DAY
I DAY
14
’ 0% 21
FIG. 9. Changes in the mean peripheral blood eosinophil (left scale; n ) and neutrophil (right scale; 0) counts, expressed as percentage of total leukocytes, at Day 21 PI in mice that received 10 L,. Each point is the mean of three mice and vertical bars represent SE of the mean.
with the exuberant tibroblast proliferation and secretion that we observed juxtaposed to the encapsulated L,. Juvenile A. simplex ES antigens have potent bioactive properties (Raybourne et al. 1983, 1986) and have been demonstrated to be released in the tissues by the invading L3 (Sakanari et al. 1988). The possibility that these ES antigens possess a fibroblast growth factor is being investigated. Fibroblast proliferation may be a host-induced protective reaction that contributes to the isolation of the penetrating L, and associated ES antigens. Recent findings indicate protease activity associated with these parasite products (Matthews 1982; Sakanari and McKerrow 1988). Peripheral blood eosinophilia has been reported to range from 4 to 41% in human anisakiasis (i.e., Sakanari el al. 1988) but was not evident in our experimental mice. We attribute this phenomena to our method of infection. Per 0s infections allow for penetration into or through the GI tract which would rapidly stimulate various immunobiologic responses of the host leading to enhanced eosinophilopoiesis. Induction of an eosinophilic response, for example, in in-
fections
with the juveniles of Trichinella the parasites to become embolized in the lungs of the host (Basten et al. 1970). Without pulmonary embolization, which may be precluded by intramuscular injection of T. spiralis juveniles rather than per OS, no eosinophilia occurs. With the surgical method of infection employed in our study, the L, showed no proclivity for the invasion of viscera. We conclude the histopathologic events in human anisakiasis and our experimental model are similar. Lesions with eosinophilic components and exuberant fibroblast proliferation and collagen secretion are common in both hosts; however, in the mouse it occurs in the absence of a peripheral blood eosinophilia. spiralis requires
ACKNOWLEDGMENTS We gratefully acknowledge Ms. Martha Stober (U.S.A., Mobile) for technical assistance. Fishes were collected by FDA inspector Mr. Dick Throm (Seattle District) with the cooperation of Mr. Mike Kinkade and the stalf at Bomstein Seafood Inc., Bellingham, Washington, and Mr. John W. McCallum and the staff at The Pacific Salmon Co., Inc., Seattle, Washington, This study was supported in part by NIH Grant AI
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19968 and American Heart Association, Alabama Affiliate, Inc., Grant 870834 awarded to S.G.K. REFERENCES ASAMI, K., AND INOSHITA, Y. 1%7. Experimental anisakiasis in guinea pigs: Factors influencing infection of larvae in the host. Japanese Journal of Parasitology 16, 415-422. BASTEN, A. B., BOYER, M. H., AND BEESON, P. B. 1970. Mechanism of eosinophilia. I. Factors affecting the eosinophil response of rats to Trichinella spiralis. Journal of Experimental Medicine 131, 12711287. BIER, J. W., DEARDORFF, T. L., JACKSON,G. J., AND RAYBOURNE, R. B. 1987. Human anisakiasis. In “Bailliere’s Clinical Tropical Medicine and Communicable Diseases: Intestinal Helminthic Infections” (Z. S. Pawlowski, Ed.), Vol. 2, pp. 723-733. Saunders, Philadelphia. BIER, J. W., AND RAYBOURNE, R. B. 1988. Anisakis simplex. (Nematoda: Ascaridoidea): Formation of immunogenic attachment caps in pigs. Proceedings of the Helminthological
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Received 5 July 1989; accepted with revision 30 October 1989
