EXPERIMENTAL

PARASITOLOGY

71,

27-38

(1990)

Trichinella spiralis: Secreted Antigen of the Infective L, Larva Localizes to the Cytoplasm and Nucleoplasm of Infected Host Cells DICKSON D. DESPOMMIER, *,I ALLEN M.GoLD,t STEPHEN W. BUCK,* VIRGINIA CAPO,S AND DAVID SILBERSTEIN§ *Division of Tropical Medicine, School of Biophysics, Columbia University, 630 West Havana, Havana, Cuba: and $Division of Medical School,

Public Health, and ?Department 168th Street, New York, New Tropical Medicine, Department Boston, Massachusetts 02167,

of Biochemistry and Molecular York 10032, U.S.A.; University of of Medicine, Harvard University U.S.A.

GOLD, A. M., BUCK, S. W., CAPO, V., AND SILBERSTEIN, D. 1990, secreted antigen of the infective L, larva localizes to the cytoplasm and nucleoplasm of infected host cells. Experimental Parasitology 71, 27-38. Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 4%50K. These antigens are stage specific for the infective first stage larva of TrichineUa spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance. 0 1990 Academic press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Trichinella spiralis; Nurse cell; Stichosome; Stichocyte; Secretory granules; 43K antigen; Ghromdtofocusing; Immunoelectrophoresis; Immunocytolocalization; Antigen deposition; Newborn larvae; Excretion-secretion (ES); Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Phosphatebuffered saline (PBS). DESPOMMIER,

Trichinelia

D. D.,

spiralis:

ture adult worms occur within the cytoplasm of a row of columnar epithelial cells in the small intestine (Wright 1979). The association of the worm with the epithelium results in the destruction of those cells harboring the parasite (Wright et al. 1987). Newborn larvae are shed by the female into the intramulticellular niche. They then migrate away from the small intestine through the blood or lymph (Harley and Gallicchico 1971; Wang and Bell 1986) and penetrate the intracellular milieu of the striated skeletal muscle cell (Despommier 1977). Within this niche, larvae undergo growth and development over the next 20

INTRODUCTION

The parasitic nematode Trichinella spiralis is unique in that it infects striated skeletal muscle cells, then induces their modulation into specialized units known as Nurse cells (Purkerson and Despommier 1974). An extensive variety of mammals worldwide is susceptible to infection (Campbell 1983) and within each host species the parasite lives most of its life intracellularly (Despommier 1983). Molting, growth, and development culminating in sexually ma’ To whom correspondence should be addressed. 27

0014-4894/90 $3.00 Copynght 0 1990 by Academic Press, Inc. All rights of reproduction in any form rcservcd.

28

DESPOMMIER

days (Despommier et al. 1975) resulting in fully infective first stage (L,) larvae (Kozek 1971). In contrast to the intestinal phase of the infection, the Nurse cell survives to sustain the parasite (Despommier 1975), often throughout the life of the host, thus ensuring the transmission of the infection from host to host through predation or scavenging. The Nurse cell is anatomically independent of the rest of the surrounding muscle tissue and functions by recruiting nutrients to itself and the parasite within, while simultaneously exporting metabolic wastes and parasite-derived products to the extracellular space (Stewart 1983). Parasiteinduced host cell transformation takes 1214 days to complete, during which time the size and number of nuclei within the infected cell increases relative to the surrounding uninfected fibers (Despommier 1975). The Nurse cells’ outer surface becomes encapsulated with collagen (Teppema et al., 1973; Ritterson 1966), and the entire parasite-host cell complex becomes surrounded by a circulatory plexus (Pagenstecher 1865; K. A. Wright, personal communication). The parasite synthesizes and stores at least 20 antigens within secretion granules in its stichocyte cells during its intracellular development within the Nurse cell (Despommier and Muller 1976; Despommier and Laccetti 1981). A few secreted antigens can, by themselves, elicit high levels of protection against challenge infection (Despommier and Laccetti 1981; Silberstein and Despommier 1984). While it is well established that each stage of T. spiralis possesses defined sets of antigens (Philipp ef al. 1980), to date none have been studied regarding its developmental regulation during Nurse cell formation. Furthermore, the biological relevance of these antigens to the parasite within the altered host cell remains unknown. In the present study, we describe the

ET AL.

temporal relationship between synthesis and secretion of a particular antigen while the worm is in its parenteral niche. We employed immunocytolocalization methods on deparaffinized sections of muscle tissue from synchronously infected mice. MATERIALSAND METHODS Infections. Trichinella spiralis infective L, larvae were maintained in male, CFW strain mice. Larvae were isolated from minced muscle tissue obtained from skinned, eviscerated carcasses by digestion for 1 hr at 37°C in 1% (w/v) pepsin (Fisher Scientific Corp, Springfield, NJ) in 1% (v/v) HCI. Worms were isolated only from mice harboring larvae that were more than 2 months old. Adult male rats (200-250 g each) were each given 5000 infective L, larvae by the oral route. On Day 7, all rats were euthanized by exposure to a pure CO, atmosphere, and the adult worms were collected from the small intestines by a thermal migration device (Despommier 1973). Newborn larvae were recovered from adult worms according to Despommier et al. (1977). Synchronous infections were initiated in 3-monthold male, CFW strain mice by injecting newborn larvae into the thigh muscles (Despommier et al. 1975). Forty thousand newborn larvae per thigh were injected into a total of six sites in each thigh. A total of 40 mice were infected. Two mice per day for 20 days were processed for immunocytolocalization. Immunocytolocalization was carried out three separate times on synchronous infections with the same results regarding the temporal relationship between antigen synthesis and secretion within the infected host muscle cell. Isolation of the 43K antigen. Excretion and secretion (ES) products of infective L, larvae were collected as described previously (Mills and Kent 1965) from lo6 worms. After concentration to a volume of ca. 9 ml on a PM-IO membrane (Amicon, Danvers, MA), the yield of protein was ca. 6 mg (Bio-Rad protein assay, Bio-Rad, Richmond, CA). This solution was dialyzed against 25 m&f piperazine-HCl buffer, pH 5.5, then subjected to chromatofocusing (Soderberg et al. 1981) on a lO-ml column (0.7 x 30 cm) of Polybuffer Exchanger-94 (Pharmacia, Inc.) equilibrated with the same buffer. This column was eluted at room temperature with piperazine buffer until the A,, fell to below 0.04. The column was then eluted with Polybuffer-74 (Pharmacia, Inc.) diluted 1:lO and adjusted to pH 3.9 with HCI. The flow rate was 16 ml/hr, and fractions of 1.0 ml were collected. Antigen of 43K appeared as a well-resolved peak at pH 4.3. Fractions were combined on the basis of their purity as judged

ANTIGEN

SPECIFIC

TO

by SDS-PAGE using reducing conditions and a discontinuous 10%/4% gel (Laemmli 1970). The silver stain of Oakley et al. (1980) was used. The protein solution was concentrated with a Centricon-10 microultrafiltration apparatus (Amicon) and purified further by HPLC using tandem 24 ml Superose-12 columns (Pharmacia, Inc.) to remove polymeric Polybuffer. The elution buffer was 50 mM ammonium bicarbonate, pH 8.2, and the flow rate was 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed by SDS-PAGE (Fig. 1). Antigen was dialyzed against 10 m&f sodium phosphate, pH 7.0, and concentrated. A yield of 0.35 mg of protein was obtained. Preparation of rabbit antibodies against the 43K antigen. Two adult New Zealand White strain rabbits each received 50 pg of purified 43K antigen in 0.2 ml of PBS injected subcutaneously in an equal volume of Freund’s complete adjuvant. Animals were bled on the first day and once a week thereafter until a strong pattern was obtained on immunoelectrophoresis. The excretion-secretion products and a defined, subcellular fraction from the infective L, larva enriched for secreted proteins served as sources of antigens used in the detection of antibodies (Despommier and Lacetti 1980). Positive serum samples were pooled and the IgG-IgA fraction was obtained by precipitation with ammonium sulfate according to the methods of Weeke (1973). Western blot analysis. Proteins and biotinylated molecular weight standards (Sigma Chemical Corp., St. Louis, MO) were subjected to SDS-PAGE using 0.25 pg of 43K antigen and 2.5 kg of the ES preparation. Electrotransfer of proteins to a sheet of nitrocellulose membrane was carried out according to Towbin et al. (1979). The membrane was incubated with a 2% solution of nonfat dry milk for 1 hr at room temperature, cut into identical halves, and developed with either the IgG-IgA fraction of rabbit serum raised against the 43K antigen or the monoclonal antibody 8A4.3.1.1 (Silberstein and Despommier 1985). Immunocytolocalization of 43K antigen. Immunocytolocalization was carried out as previously described (Capo et al. 1986). Briefly, infected thighs were removed immediately after euthanizing each mouse by cervical dislocation and placed into 10% formalin buffered with 0.01 M sodium phosphate, pH 7.0. The tissue was processed for paratIin sectioning by standard procedures employing ethanol as the dehydrating agent. Five-micrometer sections were cut and fixed onto alcohol-cleaned glass slides. At no time during processing did tissues exceed 60°C. After deparaftinization, sections were reacted with the IgG-IgA fraction of rabbit anti-43K serum or with mouse monoclonal antibody 8A4.3.1.1. Infected muscle tissue from a 6-month-old infection was used to determine the optimum concentration of rabbit anti-43K IgG-IgA. A

T. Spirdis

29

LARVAE

12

3

66-

45-

36-

24-

FIG. 1. SDS-PAGE of purified 43K antigen isolated from the infective L, larva. The gel was silver stained. Lane 1, molecular weight standards: bovine serum albumin, 66K; ovalbumin, 45K; glyceraldehyde3-phosphate dehydrogenase, 36K; carbonic anhydrase, 29K; trypsinogen, 24K. Lane 2, excretionsecretion (ES) products; lane 3, 43K antigen.

1:200 dilution of antibody gave maximum specific staining with a minimum of background staining. The IgG-IgA fraction from normal rabbit serum served as the control. Goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Dakopatts, Denmark; distributed by Accurate Chemicals, West-

30

DESPOMMIER

ET AL.

A

bury, NY) was used as the second antibody at a dilution of 1500 to detect rabbit IgG on tissue sections. Diaminobenzidine (Sigma Chemical Corp.) was employed as the enzyme substrate.

1

I

B

23456

RESULTS

The purified 43K antigen gave a single band in SDS-PAGE (Fig. 1) when developed with a silver stain (Oakley et al. 1980). This antigen was subjected to Western blot analysis, where it was compared with crude T. spiralis secretion (ES). When developed with anti-43K IgG-IgA (Fig. 2A), the pure antigen gave a single strong band with a trace of a lower M, band. ES showed a band corresponding to the 43K antigen and a pair of doublets of higher M,.. The additional bands of cross-reacting material are another antigen which has been purified and characterized as a group of closely related forms of M,45-50K. The biotinylated protein standards shown in Fig. 2 lead to M, values that are inconsistent with those calculated from unmodified standards (Fig. 1); they are useful only as reference marks to compare the two parts of Fig. 2. A more detailed description of both antigens will be published separately with evidence that the dominant epitopes of each are crossreacting N-linked carbohydrate. A similar Western blot was developed with monoclonal antibody 8A4.3.1.1 (Silberstein and Despommier 1984), which gave the same result as the anti-43K antibody (Fig. 2B). Monoclonal antibody 8A4.3.1.1 is specific for a protective “48K” antigen from T. spiralis which we believe is identical to the 43K antigen isolated in the present work. The presence of two cross-reactive antigens in crude antigen preparations was confirmed in immunoelectrophoresis experiments (not shown). Antibodies of identical specificity were elicited in both rabbits independent of how long after the initial injection of antigen the test was run and regardless of the source of antigens used for

9758-

29-

20-

14-

FIG. 2. Western blot analysis of the purified 43K antigen and the excretion-secretion (ES) products of the infective L, larva. A was reacted with rabbit IgGIgA antibodies raised against the 43K antigen, while B was reacted with mouse monoclonal antibody 8A4.3.1.1, an antibody previously shown to react with an antigen whose molecular weight was reported to be 48K. Lanes 1 and 4 contain biotinylated standards: phosphorylase b, 97,400; catalase, 58,100; alcohol dehydrogenase, 39,800; carbonic anhydrase, 29,000; trypsin inhibitor, 20,100; lysozyme, 14,300. Lanes 2 and 5 contain ES; lanes 3 and 6 contain isolated 43K antigen.

ANTIGEN

SPECIFIC

To

immunoelectrophoresis. In every case two fused arcs appeared, representing the two cross-reacting antigens. When antibodies against the 43K antigen were placed on deparaflinized sections of muscle tissue from a mouse infected for 6 months, the stichocyte cells and cuticular surfaces of the larva and the entire cytoplasm of each Nurse cell reacted positively (Fig. 3). Monoclonal antibody 8A4.3.1,1 also stained the same regions, only the intensity of the pattern was greatly reduced (data not shown). In addition, both antibody preparations reacted with the nucleoplasm, but not the nucleolus, of each nucleus in all Nurse cells (Fig. 4) and did so more intensely than with the cytoplasm. Acridine orange and Azure A-stained infected tissue confirmed that Nurse cell nu-

T. spirdis

LARVAE

31

clei and not other host components (e.g., lymphocytes, macrophages) were antigen positive (data not shown). Adjacent, noninfected muscle cells did not stain with antibody. Nonimmune rabbit IgG-IgA did not stain any host or parasite structure (Fig. 5). Synchronously infected muscle tissue revealed the following patterns of antigen distribution when reacted with the IgG-IgA fraction of rabbit anti-43K serum. No antigen was detected in the larva or host tissue prior to Day 6 postinfection. However, on Day 6 and to a greater extent on Day 7 (Fig. 6), a small region of the larva, corresponding to the anterior region of the worm, stained positively. The infected host cell did not react with antibody at this time. On Day 8, the reaction in the larva was more intense than that seen on Day 7. A

FIG. 3. Mouse muscle, infected for 6 months, was reacted with the IgG-IgA fraction of rabbit anti-43K serum. The stichocytes (S) and cuticular surface of the larva and the cytoplasm and nuclei (arrows) of the Nurse cell reacted with antibody. Adjacent noninfected tissue did not stain. Endogenous peroxidase-like activity of the red cells in capillaries (R) served as the control for the diaminobenzidine-hydrogen peroxide reaction. Longitudinal section. x250.

32

DESPOMMIER

ET AL.

FIG. 4. Same infected tissue as in Fig. 3. The Nurse cell cytoplasm (CY) and nuclei (arrows) reacted with the anti-43K antibody. The epitope(s) in the cytoplasm of the Nurse cell was distributed in a patchy pattern; the nucleoplasm portion of each nucleus contained more epitope than did the cytoplasm. Adjacent normal tissue did not react with the antibody preparation. N, nucleus; Co, collagen. Longitudinal section. x500.

few of the developing Nurse cells also had faint staining in their cytoplasm and nucleoplasm. By Day 9, all developing stichocytes and the cuticular surface of the worm and the cytoplasm and nucleoplasm of all infected Nurse cells reacted with antibody (Fig. 7). On Day 10, the intensity of staining in both parasite and infected host cells was greater than on Day 9. For the first time, antigen within the Nurse cell cytoplasm was distributed in a patchy pattern (Fig. 8). Regions of infected host cell cytoplasm adjacent to the oral end of the parasite stained most intensely, while the region furthest from the worm was more lightly stained. The uneven staining pattern increased in intensity in

both the worm and the Nurse cell up to Day 18 postinfection, maintaining the distribution noted at Day 10 (Fig. 9). DISCUSSION

Morphological studies have documented the developmental aspects of the stichosome and its complement of stichocytes during the parenteral phase of the infective L1 larva (Richeles 1955; Wu 1955; Khan 1966; Despommier 1974). In only one report was the cytoplasmic contents of the stichocytes examined at the electron microscopic level during parenteral development (Despommier 1974). A few secretory granules were observed in (Ystichocytes as early as Day 10 post-im infection with newborn

ANTIGEN

SPECIFIC

TO

T. Spirah

LARVAE

33

FIG. 5. Mouse muscle, infected for 6 months. The deparaflinized section was reacted with the IgG-IgA fraction of normal rabbit serum. No larva (L) or host component was stained. Only the endogenous peroxidase activity in red cells (R) was detected. Longitudinal section. x250.

larvae. However, the cytoplasm of the majority of both (Yand l3 stichocytes was not filled with secretory granules until Day 14. Immunocytolocalization employing immune rabbit serum from infected animals indicated that internal components of the developing larva stained positively as early as 9 days after im infection with newborn larvae (Kozek and Crandall 1974). No micrographs were presented in that study. Pritchard (1985) used whole infection serum to identify large, antigen-bearing mononuclear cells at the cyst boundary. In the present study, a secreted antigen, specific to the L, larva, localized to the nucleoplasm of all nuclei and the cytoplasm of infected transformed host cells. We employed the IgG-IgA fraction of polyvalent rabbit serum raised against the purified 43K antigen. With this reagent, we achieved strong cytolocalization. Hence, antigen was first detected in the cytoplasm of the

developing stichosome of the larva, then both inside and on the cuticular surface of the worm. Antigen was later detected within the infected host cell. These temporal events strongly suggest that the antigen is first synthesized within the developing stichocytes, then exported to the milieu of the Nurse cell for further distribution into various cellular compartments. Western blot data gave evidence that the 43K antigen shared a common epitope with a previously isolated antigen having an apparent molecular weight of 48,000 (Silberstein and Despommier 1985). Although the 43K antigen reacts with the same monoclonal antibody, 8A4.3.1 .l, as the 48K antigen, the evidence presented here does not prove that they are the same substance. The two proteins have each been shown to correspond with the most abundant component in ES in the molecular weight range above 30K (Fig. 1 in the present study, and

FIG. 6. Day 7 post-im infection with newborn larvae. The tissue was treated with the IgG-IgA fraction of rabbit anti-43K serum. Staining was only observed within the developing larva (L) and on its cuticular surface. No infected host cell component reacted positively with the antibody preparation at this time. Longitudinal section. x250.

FIG. 7. Day 9 post-im infection with newborn larvae. The same reaction conditions were used as in Fig. 6. Antigen was detected within the stichocytes and on the cuticular surface of the larva (L) and faintly throughout the cytoplasm (CY) and the nucleoplasm (arrows) of all nuclei of the infected host cell. The surrounding normal tissue did not stain. Longitudinal section. x250. 34

ANTIGEN

SPECIFIC TO T. qIirU&

LARVAE

35

FIG. 8. Day 11 post-im infection with newborn larvae. The same general pattern of staining was obtained at this time point as seen at Day 9 (Fig. 7). However, the intensity of the reaction was greater than previously noted. Patches of heavy antigen deposition, usually adjacent to the anterior portion of the worm, were also characteristic for this time point. Normal tissue did not react with antibody. L, larva, N, nucleus. Longitudinal section. x250.

Fig. 9 in Silberstein, pp. 81-82). We believe that the discrepancy in apparent molecular weights results from differences in the way the antigens were prepared for SDS-PAGE: the 43K antigen and protein standards were reduced before electrophoresis, while the 48K antigen and standards were not. Additional data that strongly support the identity of the two proteins will be presented in a forthcoming manuscript. The conclusions presented here do not depend in any way upon the identity or nonidentity of these two substances. Since the Western blot pattern of the 43K antigen showed that our antiserum recognizes a second antigen (45-50K) in the secretions of the larva, it is still unclear which antigen(s) in this group is associated with host cellular components. Furthermore, the specific cellular constituents within the

Nurse cell that interacted with antigen could not be determined from our results. The induction phase for Nurse cell formation probably occurs during the first 1 or 2 days after the newborn larva enters the muscle cell. The growth curve of the larva (Despommier et al. 1975) and the observations of infected cells from synchronous infections by light and electron microscopy have provided evidence in support of this view (Despommier 1975; Stewart 1983). Since we could not detect the 43K antigen during the earliest time points (i.e., Days l-5), we suggest that it is likely that this antigen is not involved in the induction of Nurse cell formation. Alternatively, it is possible that with more sensitive techniques (e.g., autoradiography with labeled antibodies) antigen might be detected in the parasite and infected host cell earlier in the

36

DESPOMMIER

ET AL.

FIG. 9. Day 18 post-im infection with newborn larvae. The intensity of the staining reaction was maximum at this time point. L, larva; N, nucleus. Longitudinal section. X250.

infection. This possibility is currently under study. During the first 10 days of infection, the invaded muscle cell nuclei enlarge and become randomly distributed throughout the disorganized cytoplasm (Despommier 1975; unpublished observations). However, nuclei have not been noted in increased numbers within the infected cell until well after the sixth day. Hence, it is possible that the 43K antigen is involved in this process. The vascular plexus which surrounds the maturing Nurse cell (Pagenstecher 1865) and the collagenous outer layer (Teppema et al. 1973) are both present only later on in the infection. Hence, collagen synthesis and angiogenesis are two processes which coincide with the presence of antigen in the nuclei of the Nurse cell. The long-term association of antigen with the cytoplasm and nucleoplasm of the Nurse cell suggests that continued low levels of

antigen secretion occur throughout infection. It is plausible that this antigen is involved in maintaining the redifferentiated state of the infected muscle cell. The occurrence of foreign proteins within the nucleus of infected host cells has been documented for a variety of intracellular infectious agents, particularly for the herpesviruses (Roizman 1982; Kops and Knipe 1988) and adenoviruses (Ginsberg 1984). In a few cases, their functions have been intimated but not definitively determined. For example, adenoviruses produce proteins which interfere with host cell DNA and protein synthesis (Levine and Ginsberg 1968; Babiss and Ginsberg 1984), but, as with the herpesviruses, the mechanisms are not fully understood. Rickettsiae, all related to Rickettsia rickettsiae (Wisseman 1981), occasionally infect host cell nuclei, but association of rickettsial proteins with host cell DNA or nucleoproteins has not

ANTIGEN

SPECIFIC TO T. Spiralis LARVAE

been documented. In this latter instance, the rickettsial organism causes the death of its host cell. Leishmania, Plasmodia, Trypanosoma cruzi, and Toxoplasma gondii are examples of eukaryotic intracellular parasites, infection with which results in the eventual death of the host cell. T. spiralis is one of a limited number of examples of intracellular infectious agents which have evolved a survival strategy in which an exquisitely balanced life style is achieved by keeping alive the cell in which it lives. We speculate that in the case of T. spirulis this balance is maintained by the interaction of secreted parasite products with constituents of the cytoplasm and the nucleus of the infected muscle cell. Determining the structures and functions of the secreted antigens of L, larvae will help resolve whether or not any are critical for the induction and maintenance of the Nurse cell.

tion device for the rapid collection of large numbers of intestinal helminths. Journal of Parasitology 59, 933-93s.

DESPOMMIER, D. D. 1974. The stichocyte of Trichinella spiralis during morphogenesis in the small intestine of the rat. In “Trichinellosis” (C. Kim, Ed.), pp. 239-254. Intext, New York. DESPOMMIER, D. D. 1975. Adaptive changes in muscle fibers infected with Trichinella spiralis. American Journal

of Pathology

78, 477-496.

DESPOMMIER, D. D. 1983. Biology. In “Trichinella and Trichinosis” (W. C. Campbell, Ed.), pp. 75151. Plenum, New York/London. DESPOMMIER, D., ARON, L., AND TURGEON, L. 1975. Trichinella spiralis: Growth of the intracellular muscle larva. Experimental Parasitology 37, 108-l 16. DESPOMMIER, D. D., CAMPBELL, W. C., AND BLAIR, L. S. 1977. The in vitro and in vivo analysis of immunity to Trichinella spiralis in mice and rats. Parasitology 74, 109-119. DESPOMMIER, D. D., AND LACCETTI, A. 1981. Trichinella spiralis: Proteins and antigens isolated from a large-particle fraction derived from muscle larva. Experimental Parasitology 51, 279-295. DESPOMMIER, D. D., AND MULLER, M. 1976. The stichosome and its function in the mature muscle larva of Trichinella spiralis. Journal of Parasitology 62, 775-785.

ACKNOWLEDGMENTS We thank Lex van der Ploeg, David Misek, Ramona Polvere, and Alice Baruch for their helpful discussions and critical review of our data and Mrs. Terri Terilli for skillfully typing the manuscript. We extend a special thanks to our audio-visual department for faithfully reproducing the informational contents of each of our numerous photomicrographs and gels contained herein. This research was supported, in part, by NIH Grant ROl-AI-10627. REFERENCES ALI KHAN, J. E. 1966. The post-embryonic development of Trichinellu spiralis with special reference to ecdysis. Journal of Parasitology 52, 248-259. BAEIISS, L. E., AND GINSBERG, H. S. 1984. Adenovirus type 5 early region lb gene product is required for efficient shutoff of host protein synthesis. Journal of Virology 50, 202-212. CAMPBELL, W. C. 1983. Modes of transmission. In “Trichinella and Trichinosis” (W. C. Campbell, Ed.), pp. 425-444. Plenum, New York/London. CAPO, V., SILBERSTEIN, D., AND DESPOMMIER, D. D. 1986. Immunocytolocalization of two protectioninducing antigens of Trichinella spiralis during its enteral phase in immune and nonimmune mice. Journal of Parasitology 72,931-938. DESFWMMIER, D. D. 1973. A circular

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GAMBLE, H. R. 1985. Trichinella spiralis: Immunization of mice using monoclonal antibody affinityisolated antigens. Experimental Parasitology 59, 398-404.

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MILLS, C. K., AND KENT, N. H. 1965. Excretions and secretions of Trichinella spiralis and their role in immunity. Experimental Parasitology 16, 300-310. OAKLEY, B. R., KIRSCH, D. R., AND MORRIS, N. R. 198Q. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry

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STEWART, G. 1983. Pathophysiology of the muscle phase. In “Trichinella and Trichinosis” (W. C. Campbell, Ed.), pp. 241-264. Plenum, New York/ London. TEPPEMA, J. S., ROBINSON, J. E., AND RUITENBERG, E. J. 1973. Ultrastructural aspects of capsule formation in Trichinella spiralis infection in the rat. Parasitology 66, 291-296. TOWBIN, H., STAEHELIN, T., AND GORDON, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National

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Received 3 April 1989; accepted with revision 8 December 1989