Anisakis Simplex (Nematoda: Ascaridoidea): Formation of Immunogenic Attachment Caps in Pigs

Proc. Helminthol. Soc. Wash. 55(1), 1988, pp. 91-94

Research Note Anisakis simplex (Nematoda: Ascaridoidea): Formation of Immunogenic Attachment Caps in Pigs

JEFFREY W. BIER AND RICHARD B. RAYBOURNE Division of Microbiology, Food and Drug Administration, Washington, D.C. 20204

ABSTRACT: Anisakis simplex larvae maintained in runs (indoor winter heat), fed USDA ration 1160 simple salt solutions were observed attached to the at 25 g/kg/day, and allowed water ad libitum. surface of a petri dish in vitro by a translucent cap. The pigs were treated with the anthelmintic At- Previous reports attributed the formation of these caps to contributions by human serum. Dissolved caps re- gard (Fermenta Animal Health Products, St. acted in Western blot with the sera of rabbits sensitized Louis, Missouri) at least 2 wk before experimen- with A simplex excretory-secretory products. Caps were tal exposure. Third-stage larvae of A. simplex demonstrated in vivo in the mucosae of experimentally were isolated from the viscera of bocaccio (Se- infected miniature swine; they were partially composed of larval excretory-secretory products and remained bastes paucispinis) by digestion with 2% pepsin- embedded in stomach mucosae after removal of the HC1 (pH 2) at 35°C. Larvae were maintained at invasive larvae. 2-6°C before pigs were experimentally infected. KEY WORDS: Nematoda, Ascaridoidea, anisakiasis, Food was withheld from the pigs for 24 hr before Anisakis simplex, nematode larvae, attachment cap, exposure. Each pig was then fed 100 larvae by calotte, excretory-secretory products, immunogenic, miniature swine, Sus scrofa, stomach mucosae. stomach tube. Pigs were necropsied 24 hr after exposure; tissues were fixed in Bouin's fluid, de- Formation of a calotte or cap by approxi- hydrated, and sectioned by the method of Hinton mately 16% of Anisakis simplex (=A. marina) etal. (1987). larvae isolated from herring and incubated in Rabbit antiserum to larval excretory-secretory tubes of human serum at 37°C was described by (ES) proteins was produced by subcutaneous in- van Thiel (1967). On the basis of acrolein-Schiff jection of 60 yug ES protein in Freund's complete staining, the clear cap was identified as consisting adjuvant, followed in 2 wk by 60 Mg ES protein of albumin (P. van Duyn in van Theil, 1967). in Freund's incomplete adjuvant. Control serum The cap was found with sera from persons un- was obtained from the rabbits before immuni- likely to have experienced any exposure to ani- zation. ES proteins used for immunization were sakine nematodes and with the sera of anisakiasis collected as described previously (Raybourne et patients. Noting that parasite excretory products al., 1983). were granular, van Thiel postulated that the clear Sections cut at 6-10 ^m thickness were rehy- cap was at least partly of host origin. We have drated in PBS and incubated on glass slides with frequently observed the formation of similar a 1:40 dilution of rabbit anti-ES serum or control structures in vitro in simple salt solutions such serum for 1 hr at room temperature. The sections as phosphate-buffered saline (PBS) or Ringer's were washed for 30 min in 3 changes of PBS and solution and in more complex media such as incubated with a 1:40 dilution of fluorescein- Medium 199 (with and without 10% fetal bovine labeled goat anti-rabbit IgG (Sigma Chemical Co., serum). This observation suggested that struc- St. Louis, Missouri) for 1 hr. The sections were tures similar to van Thiel's "calotte" could be rinsed in PBS, mounted in 90% glycerol-10% formed by parasite products alone and raised the PBS, and examined with a Leitz fluorescence mi- question as to whether formation of these struc- croscope. Photographs were taken with an au- tures accompanied attachment to and invasion tomatic metering device designed to provide a of stomach mucosae by A. simplex larvae. uniform level of exposure (Wild, Heerbrugg, In this report, culture conditions were the same Switzerland). as those described previously (Raybourne et al., Attachment caps adhering to the bottom of a 1986), in which PBS (pH 7.2) with 1% glucose petri dish were rinsed 3 times with PBS and made was used. For experimental exposure, Hormel- soluble by heating in PBS with 10% sodium do- Hanford miniature pigs (Sus scrofa} were main- decyl sulfate (SDS) and 5% 2-mercaptoethanol. tained in masonry kennels with indoor-outdoor The samples were separated on a 12% SDS-PAGE 91

Figures 1-4. Longitudinal sections through attachment caps of Anisakis simplex larvae in fundus of pig stomach. Scale bars = 0.1 mm. 1. Anterior end, 3rd-stage larva stained by modified Shorr's stain after Vetterling and Thompson (1972). Note that cationic sites in attachment cap are deeply stained by Biebrich scarlet stain (bright orange area surrounding anterior end) and differentiated from the adjacent red blood cells (rust colored) stained by orange G. 2. Lesion showing attachment cap from which nematode was physically removed. Similar structures are encountered in mucosae of infected pigs. Azure A-Eosin B stain after Lillie (1965). 3. IFA staining of attachment site with rabbit antiserum to Anisakis simplex and fluoresceinated goat antibody to rabbit serum. Exposure time 30 sec. 4. Same attachment site as in Figure 3 (5 intervening sections) treated with presensitization serum and goat fluoresceinated antibody to rabbit antibody. Exposure time 2 min 30 sec. Exposure demonstrates yellow autofluorescent material and background green fluorescene associated with IFA staining.

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 55, NUMBER 1, JANUARY 1988 93 gel, electroblotted onto a nitrocellulose mem- brane, and reacted with a 1:50 dilution of anti- 180 ES serum or control serum (Raybourne et al., 116 1986). Bands were developed with protein A per- oxidase (Boehringer Mannheim, Indianapolis, 84 Indiana) diluted 1:1,000. One lane of the gel/blot 58 contained 10 /ug ES material. 48-5 When crowded (> 100 larvae per 75-mm petri dish), a greater proportion of the worms appeared to form caps than when fewer organisms 36-5 were present (< 50 larvae per petri dish). Grossly, 26 all the larvae in a culture appeared to be attached to the petri dish by their anterior ends. Individual nematodes were extracted by pulling with for- ceps, leaving the cap attached to the dish. Caps of nematodes that broke were not used. The caps, or groups of caps, were translucent and refractile, 1 2 and could be scraped from the petri dish. They Figure 5. Comparison by Western blot of antigens appeared to be similar to the structures described from solubilized attachment caps and ES products col- by van Thiel (1967) which formed in the pres- lected from in vitro cultures ofAnisakis simplex. Lanes: ence of, and were thought to be partially com- 1 and 3, solubilized cap; 2 and 4, ES proteins. Lanes 1 posed of, normal human serum. The formation and 2 were incubated with anti-ES serum; lanes 3 and 4 were incubated with control serum. Arrows indicate of caps by A. simplex larvae in media without antigens of similar molecular weight present in solu- serum or other protein additives suggests that bilized caps and ES proteins. caps may be formed entirely from material of parasite origin. Similar structures apparently form in the (Raybourne et al., 1986). These caps may pro- stomach mucosae of experimentally infected pigs. vide a residual of antigenic material that may In vivo, the cap surrounds the rudimentary lips prolong the inflammatory response observed af- and anterior end of the stage 3 larvae (Fig. 1). ter nematodes can no longer be detected in chronic Caps were also observed around the lips and human anisakiasis, or after surgical removal of anterior ends of A. simplex stage 4 larvae in pigs. the larva (Oshima, 1972). The flaring shape and knoblike handle at the apex The mechanism by which the caps are formed suggested that the cap aids the parasite in main- is not known. Proteolytic enzymes present in the taining attachment to the stomach mucosae. anterior secretory structures and ES products of Some components of these structures remained A. simplex larvae (Ruitenberg and Londersloot, at attachment sites that the worms vacated or 1971; Mathews, 1982, 1984) may be involved. from which they were removed mechanically (Fig. These proteases may contribute to the formation 2). Caps with nematodes are frequently observed of attachment caps by a mechanism similar to in tissue sections of infected pigs. Caps were ob- that of the proteases which function to form a served in more than 25 lesions with worms that gel in the limulus amoebocyte lysate assay (Shi- were sectioned, stained, and examined in detail. shikuraet al., 1983). At least some of the components of the cap We thank Roger Mathews and the animal han- were of parasite origin, as demonstrated by in- dlers at the Beltsville Research Facility of the direct fluorescent antibody (IFA) staining of the Food and Drug Administration for their help in cap in vivo with anti-ES serum (Figs. 3, 4). Al- handling and caring for the pigs. though the cap proteins could not be resolved on SDS gels as clearly as were the ES proteins, some Literature Cited of the components of the caps formed in vitro Hinton, D. M., J. J. Jesop, A. Arnold, R. Albert, and appeared to be equivalent to parasite ES pro- F. A. Hines. 1987. Evaluation of immunotox- teins, based on their reactivity in Western blots icity in a subchronic study of triphenyl phosphate. Toxicology and Industrial Health 3:71-89. (Fig. 5). These ES proteins, present in the su- Lillie, R. D. 1965. Histopathologic Technic and Prac- pernatant fluid of larval maintenance cultures, tical Histochemistry. McGraw-Hill Book Co., New are important immunogens in human anisakiasis York.

Mathews, B. 1982. Behaviour and enzyme release by of mitogen-induced rodent lymphoblast prolifer- Anisakis sp. larvae (Nematoda: Ascaridida). Jour- ation. Experimental Parasitology 55:289-298. nal of Helminthology 56:177-183. Ruitenberg, E. J., and H. J. Londersloot. 1971. His- . 1984. The source, release and specificity of tochemical properties of the excretory organ of proteolytic enzyme activity produced by Anisakis Anisakis sp. larva. Journal of Parasitology 57:1149- simplex larvae in vitro. Journal of Helminthology 1150. 58:175-185. Shishikura, F., S. Nakamura, K. Takahashi, and K. Oshima, T. 1972. Anisakiasis. Pages 301-399 in K. Sekiguchi. 1983. Coagulogens from four living Morista, Y. Komioya, and H. Matsubayashi, eds. species of horseshoe crabs (Limulidae): compari- Progress of Medical Parasitology in Japan. Vol. 4. son of their biochemical and immunochemical Meguro Parasitological Museum, Tokyo. properties. Journal of Biochemistry 94:1279-1287. Raybourne, R., T. L. Deardorff, and J. W. Bier. 1986. van Thiel, P. H. 1967. Le phenomene de la "for- Anisakis simplex: larval excretory-secretory pro- mation de la calotte" chez la larve d''Anisakis ma- tein production and cytostatic action in mam- rina (Linnaeus, 1767) dans du serum humain. An- malian cell cultures. Experimental Parasitology 62: nales de Parasitologie Humaine et Comparee 42: 92-97. 633-642. , R. S. Desowitz, M. M. Klicks, and T. L. Dear- Vetterling, J. M., and D. E. Thompson. 1972. A poly- dorff. 1983. Anisakis simplex and Terranova sp.: chromatic stain for use in parasitology. Stain inhibition by larval excretory-secretory products Technology 47:164-165.

Proc. Helminthol. Soc. Wash. 55(1), 1988, pp. 94-96

Research Note Binding of Concanavalin A to Areas Compatible with the Locations of the Amphids and Phasmids of Larvae of Dirofilaria immitis (Nematoda: Filarioidea) and Toxocara canis (Nematoda: Ascaridoidea)

DWIGHT D. BOWMAN,1 DAVID ABRAHAM,2 MARCIA MlKA-GRIEVE,3 AND ROBERT B. GRIEVED Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706

ABSTRACT: Third-stage and 4th-stage larvae of Di- amination, each larva was found to have 2 bright areas rofilaria immitis and infective-stage larvae of Toxocara of fluorescence on both the anterior and posterior ends; canis were fixed in 10% formalin and incubated with these areas of fluorescence were not present on worms the fluorescein-labeled lectin, concanavalin A. The lar- that had been incubated with fluorescein-labeled con- vae were then washed, mounted on slides, and ex- canavalin A that had been preincubated with methyl amined using an epifluorescent microscope. Upon ex- a-D-mannopyranoside. The locations of the areas of fluorescence on these worms were found to be consis- tent with the described locations of the amphids and phasmids of these larval stages. 1 Present address: Department of Microbiology, Im- munology & Parasitology, New York State College of KEY WORDS: concanavalin A, amphids, phasmids, Veterinary Medicine, Cornell University, Ithaca, New Dirofilaria immitis, Toxocara canis, Nematoda, Filar- York 14853. ioidea, Ascaridoidea, lectins, chemoreception, cuticle, morphology. 2 Present address: Department of Microbiology, Thomas Jefferson University, Philadelphia, Pennsyl- As part of a study on various surface charac- vania 19151. teristics of larval nematodes, 3rd- and 4th-stage 3 Present address: Department of Pathology, Colo- rado State University, Fort Collins, Colorado 80523. larvae of Dirofilaria immitis and infective larvae 4 To whom correspondence should be addressed. of Toxocara canis were incubated with the fluo-