Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity

EXPERIMENTAL

PARASITOLOGY

50,

103~ 114 (1980)

Fasciola hepatica: Glycocalyx Replacement in the Juvenile as a Possible Mechanism for Protection against Host Immunity R. E. B. HANNA Zoology

Department,

Queen’s

(Accepted

University,

Belfast BT7 INN,

for publication

2 November

Northern

Ireland

1979)

HANNA, R. E. B. 1980. Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity. Experimental Parmitofogy 50, 103-114. The response of newly excysted juvenile Fasciola hepafica to immune sheep serum under in vitro conditions was examined using indirect fluorescent antibody labeling and electron microscopy. Flukes acquired a continuous layer of host IgG over the surface during incubation in the presence of antiserum, but when transferred to a medium lacking antiserum they actively sloughed this layer and replaced the former glycocalyx, by a new antigenically similar surface coat. Electron microscope examination of juvenile flukes verified than an immune complex formed at and sloughed from the tegumental surface of those which were incubated in immune serum. TO secretory bodies produced by the GER/Golgi system of the tegumental cells and stored in the metacercariae were discharged at the apical surface of the tegument. possibly in response to antibody binding. When cycloheximide was included with immune serum in the incubation medium the tegumental cells were unable to synthesize new TO bodies to replace losses and the number of TO bodies decreased so that the cytoplasm of the tegumental cells and surface syncytium became virtually devoid of TO bodies within 48 hr. INDEX DESCRIPTORS: Fasciola hepatica; Trematode; In vitro excysted juveniles; Tegument; Glycocalyx replacement; Immune protection; Immunoglobulin; Protein synthesis; Exocrine secretion; Indirect fluorescent antibody technique; Electron microscopy: Cycloheximide.

INTRODUCTION

It is well established that the presence of the juvenile invading stages of Fasciola spp. engenders a significant immunological response in the host. The titer of specific antibody in the bloodstream of sheep and cattle reaches a maximum during the first 6 weeks of infection but declines once the flukes are established in the bile ducts (Movsesijan and Jovanovic 1975; Hanna and Jura 1977). Apparently the production of antibodies has little protective significance to the host, at least during a primary infection. The flukes survive peak antibody titers while in the liver parenchyma and can exist for extended periods of time in the bile ducts of the natural host. Sheep appear unable to resist secondary and subsequent infections

with Fasciola hepatica (reviews by Dawes and Hughes 1964, 1970; Smithers 1976) but it is clear that infected cattle (Kendall 1967; Ross 1967), rats (Hayes et al. 1972), and mice (Lang 1967) can at least partially destroy a challenge infection. The exact mechanism by which flukes of a challenge infection are killed is at present uncertain (Smithers 1976), but it is likely that specific humoral and cell-mediated factors both act in the destruction of the parasites (Armour and Dargie 1974). Recent evidence suggests that immune destruction of some helminth parasites involves the eosinophil as effector in an antibodydependent system (Butterworth 1977). During a primary infection, Fasciola spp. can evade the host response. Such activity has been intensively studied in Schisto103 0014~4894/8O/o4O103-12$02.OO/O Copyright 0 1980 by Academic Press. Inc. AI1 rights of reproduction in any form reserved.

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soma where several mechanisms may oper-

ate to protect the adult worms. Absorption of host antigens on to the surface may mask them from immunological recognition (Smithers et al. 1969). It also seems that the heptalaminate surface may be capable of rapid renewal when under immune attack (Perez and Terry 1973; Kusel et al. 1975; Wilson and Barnes 1977). Using worm transfer techniques, Hughes and Harness (1973a, b) were unable to demonstrate a “host-antigen disguise” phenomenon in juvenile and adult Fasciola hepatica. Possibly the young fluke burrowing in the liver is constantly moving out of range of the cellular effector mechanisms (Dawes and Hughes 1964) while the adult is shielded from immune attack once it enters the bile ducts (Lang 1967). Goose (1978) showed that certain excretory or secretory products of Fasciola hepatica collected during dry incubation of the flukes in polythene tubes were toxic to immunocompetent cells of the host when tested in vitro. It is probable that the active principle in these exudates was proteolytic enzyme from the gut ceca. The tegumental surface of invading flukes is continually exposed to the host, so it is likely that an immunological attack is directed against the components of this surface. Indeed, high titers of antibody specific for the surface of Fasciola gigantica metacercariae were detected in experimentally infected calves (Hanna and Jura 1977). Thus it might be expected that a mechanism for evading the host’s immunological responses would be centered in the tegument of Fasciola. The ultrastructure of the tegument of adult flukes has been known for some time (Threadgold 1963, 1967) and Bennett and Threadgold (1975) have described certain morphological changes which occur during the early stages of infection in mice. However, little attempt has been made to designate specific physiological functions to the tegument. In particular, the role of the vari-

ous secretory bodies produced by GER/ Golgi activity in the tegumental cells of Fasciola and most other flukes and tapeworms studied (Lee 1966, 1972) remains largely undetermined. Autoradiographic studies have indicated that Golgiderived vesicles give rise to the surface membrane and the glycocalyx (Oaks and Lumsden 1971; Hanna 1980). In view of the fact that the glycocalyx of parasitic platyhelminths lies at the interface with the host, it is probable that this region has considerable importance in the host-parasite relationship (Lumsden 1975). The morphology and histochemistry of the glycocalyx of Fasciola hepatica has been precisely defined by Threadgold (1976). Work is now in progress to examine in detail the immunological role of the tegument of Fasciola hepatica using immunofluorescence microscopy and related techniques. In the studies reported here the response of living juvenile flukes to the presence of specific antibody adhering to the tegumental surface was examined at light and electron microscope levels. METHODS

AND MATERIALS

Antiserum. Six cross-bred lambs were each infected orally with 200 metacercariae of Fasciola hepatica which were obtained from snails (Lymnaea truncatula) reared and infected in the laboratory. Serum was collected from each lamb prior to infection, at weekly intervals during the first 8 weeks of infection, and at fortnightly intervals thereafter until the infection was 30 weeks old. At autopsy each animal was found to harbor .50- 100 mature flukes in the bile ducts. The concentration of specific antibody was monitored throughout the infection period for each animal using the indirect fluorescent antibody (IFA) technique described by Hanna and Jura (1977). Since the highest antibody titers were recorded from the serum samples collected 6 weeks after infection, these were used as

Fasciola

hepaticn:

GLYCOCALYX

the source of immune serum in the following experiments. Juvenile flukes. Laboratory-reared metacercariae were scraped from the cellophane strips on which they were stored and were incubated in 0.5% pepsin in Hanks’ saline (pH 2, 37 C for 20 min) to remove the outer cyst walls. After washing with Hanks’ saline (pH 7.2), the metacercariae were incubated at 37 C in excystment medium. This was prepared immediately before use by mixing 5 ml each of N/20 HCl and 0.8% NaCl + 1.O% NaHCO, and adding sodium tauroglycocholate (50 mg) and L-cysteine-HCl(40 mg). Newly excysted juvenile flukes were removed from the excystment medium after 2 hr and rinsed in warm tissue culture medium NCTC 135 prior to use. Experiment 1. Newly excysted juvenile flukes were incubated at 37 C for 30 min in NCTC 135 containing 10% immune serum. The medium was then removed and the flukes were quickly rinsed three times in warm NCTC 135. Flukes, 50-100, were immediately fixed in 10% neutral formalin for 5 min, transferred to phosphatebuffered saline (PBS, pH 7.2), and stored at 4 C. The remaining flukes were incubated at 37 C in NCTC 135 with or without 10% preinfection sheep serum and samples of 50-100 flukes were removed, fixed and stored at intervals up to 5 h. When incubation was complete the flukes were rinsed in several changes of PBS before being labeled with fluorescein-conjugated rabbit anti-sheep immunoglobulin (Wellcome Reagents Ltd., Beckenham, England) (diluted 1:40 with PBS) for 30 min at room temperature . After rinsing, the labeled flukes were mounted in buffered glycerol (9 ml glycerol + 1 ml PBS) and examined using a Zeiss 2 Fl reflected fluorescence microscope. The extent of surface labeling on each fluke was scored as follows: 3 entire surface labeled 2 approximately 2/3 surface labeled

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1 approximately l/3 surface labeled 0 surface unlabeled. The score for each batch of flukes was determined by totaling the individual scores, and this was expressed as a percentage of the maximum possible score for that batch of flukes, i.e., (number of flukes) x 3, thus, Percentage surface label = total score (number of flukes) x 3

x 100.

Control incubations were carried out using flukes fixed in 10% neutral formalin before or after exposure to 10% immune serum. Some flukes were removed after 3 -5 hr of incubation in NCTC 135 and reexposed to 10% immune serum before fixation and labeling with conjugate. In order to test that fluorescent labeling did indeed represent an immunological reaction some flukes which had been incubated in immune serum were treated with unconjugated rabbit anti-sheep immunoglobulin (blocking serum) prior to labeling with the fluorescein conjugate. The experiments were carried out six times using serum from a different lamb on each occasion. Experiment 2. Newly excysted flukes were incubated at 37 C for up to 48 hr in NCTC 135 containing 10% immune or preinfection sheep serum and 1 mM cycloheximide, which is known to inhibit protein synthesis in fluke tissues (Hanna and Threadgold 1976). Corresponding incubations were carried out in media lacking cycloheximide. Batches of flukes were removed at intervals up to 48 hr, rinsed in Hanks’ saline and fixed with 4% Millonig-buffered glutaraldehyde containing 3% sucrose. After 24 hr fixation at 4 C the flukes were washed thoroughly with Millonig buffer + 3% sucrose, postfixed in 1% Millonig-

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Fasciola hepaticu: GLYCOCALYX

buffered osmium tetroxide, dehydrated through alcohol and propylene oxide, and embedded in Araldite embedding resin. Ultrathin sections were cut from each block using a Reichert OM U2 ultrotome, mounted on copper grids, double stained with uranyl acetate and lead citrate, and examined in an AEI EM 801 electron microscope. RESULTS

Experiment I All juvenile flukes which were fixed immediately after treatment with 10% immune serum and subsequently exposed to conjugated anti-sheep immunoglobulin showed bright fluorescent labeling over the entire surface. The labeling was disposed in a characteristic reticular pattern which was particularly well developed over the anterior end (Fig. 1). Flukes which were incubated in NCTC 135 with or without nonimmune serum, following treatment with immune serum, showed incomplete surface labeling when subsequently exposed to conjugate. The proportion of the surface which was labeled depended on the length of the incubation period. Thus, most flukes which had been incubated for M hr after removal from the immune serum had about two-thirds of their surface labeled, the sheep IgG having been lost from a wide crescentic area anterior to the ventral sucker (Fig. 2).

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Less than one-third of the surface of those flukes incubated for 1M hr still possessed adherent sheep IgG, while with still longer incubation immunoglobulin was lost from all parts of the surface except for small patches around the oral sucker and excretory pore (Fig. 3). By 5 hr the surface of all the flukes was virtually clear of adherent immunoglobulin. In control experiments, where the flukes were fixed before or immediately after exposure to immune serum and subsequently incubated in NCTC 135 with or without nonimmune serum, the surface of all flukes remained completely coated with sheep IgG throughout the 5-hr period. When flukes which had been given 3-5 hr incubation in NCTC 135 were reexposed to 10% immune serum their surfaces immediately became completely covered again with adherent sheep IgG. Quantitative estimates of the percentage of surface bearing sheep IgG after various periods of incubation in NCTC 135 following exposure to immune serum are presented in Fig. 4. It is apparent that adherent IgG was lost almost exponentially during the incubation by living flukes, while no such loss occurred with the fixed controls. Flukes treated with a blocking serum prior to labeling with fluorescein conjugate were always completely negative in the IFA test showing that fluorescent labeling resulted from a true immunological reaction.

FIG. 1. Fusciola hepatica, newly excysted in vitro. Treated with 10% immune serum from sheep and exposed immediately thereafter to fluorescein-conjugated anti-sheep immunoglobulin. Fluorescent labeling is disposed over the surface in a reticular pattern with is particularly evident anteriorly between the oral sucker (0) and the ventral sucker (V). x 1250. FIG. 2. Fasciola hepaticn, newly excysted in vitro. Treated with 10% immune serum from sheep, then incubated in NCTC135 with 10% nonimmune serum for 30 min prior to labeling with fluoresceinconjugated anti-sheep immunoglobulin. The adherent immunoglobulin is being lost from the surface. Note particularly the unlabeled area anterior to the ventral sucker (V). x 1250. FIG. 3. Fusciola hepatica, newly excysted in vitro. Treated with 10% immune serum from sheep, then incubated in NCTC 135 with 10% nonimmune serum for 3 hr prior to labeling with fluoresceinconjugated anti-sheep immunoglobulin. The surface is almost clear of adherent immunoglobulin, small patches remaining only around the oral sucker (0) and excretory pore (E). Strongly autofluorescent concretions (EC) in the excretory system are apparent in the region of the ventral sucker. x 1500.

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A thin continuous dense glycocalyx was apparent on the outer aspect of the apical membrane of the tegument. TO bodies were observed at the surface in various stages of exocrine discharge. Those whose contents had been exuded appeared flattened, and , relabeled often lay in a tangential orientation relative I to the surface. Their bounding membrane I I was sometimes seen to be continuous with I I the plasmalemma (Fig. 6). Flukes which had been incubated for 6 or 12 hr in the presence of immune serum had large amounts of flocculent material loosely OJ 1 r attached to the surface and numerous TO ‘ 3 5 I 0 2 Hours 'Chase bodies were present in the surface synFIG. 4. Graph showing loss of immune complex cytium and at the apical surface (Fig. 7). from the surface of juvenile Fax&la hepatica treated Many of the tegumental cells were less with sheep antiserum and subsequently incubated in densely packed with TO bodies than equiv“chase” medium lacking antibody. alent controls. This difference was increasingly obvious after longer incubation, especially in the presence of cycloheximide Experiment 2 (Fig. 8). Occasionally membrane-bound The tegument of juvenile flukes incu- “blebs” of the apical cytoplasm were seen bated for up to 48 hr in 10% nonimmune detached from the surface (Fig. 7). After 48 hr incubation in the presence of sheep serum generally showed little change from the preincubation condition although immune serum and cycloheximide most of dense TO secretory bodies were more the tegumental cells in all flukes examined abundant in the surface syncytium. Most of presented a shrunken appearance, the TO the tegumental cells retained large numbers bodies having virtually disappeared (Fig. 9). of TO bodies no matter what the incubation Frequently areas of the surface synperiod (Fig. 5) and no significant differ- cytium were also devoid of TO bodies and ences were noted between the cyclo- were much thinner and flatter than the equivalent controls. Surface blebbing was heximide-treated and untreated flukes.

--L

FIG. 5. Fasciola hepafica excysted in tjitro. Surface syncytium (S) and type 0 tegumental cells of a control fluke incubated for 24 hr in NCTC 135 with 10% nonimmune sheep serum and 1 mM cycloheximide. The appearance is similar to that of unincubated material. TO secretory bodies pack the tegumental cells and are accumulating in the surface syncytium where some are seen discharging at the apical surface (A). The nucleus of a tegumental cell (N) and surface spines (Sp) are evident. x 15,750. FIG. 6. Fasciola hrparica excysted in vifro. Apical surface of tegument of a control fluke incubated for 24 hr in NCTC 135 with 10% nonimmune sheep serum. Note the dense glycocalyx (G) lining the outer aspect of the apical plasma membrane (M). TO bodies exude their contents into the glycocalyx by exocytosis and become flattened in the process (arrows). ~37,800. FIG. 7. Fasciola heparica excysted in vitro. Surface syncytium (S) and tegumental cells of a fluke incubated for 12 hr in the presence of 10% immune sheep serum and 1 mM cycloheximide. Note the large amount of flocculent immune complex (F) attached to the surface. Numerous TO bodies occur in the surface syncytium (S) and discharge at the apical membrane (M), but fewer occur in the tegumental cell cytoplasm (TC) than is the case with equivalent controls. Membrane-bound portions of cytoplasm appear to be blebbing from the surface (B). Nuclei (N) and mitochondria (Mi) of the tegumental cells are evident. x 15,750.

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to antiserum, the adherent layer of immunoglobulin remained intact throughout subsequent “chase” incubation. Thus, it appears that the juvenile flukes actively sloughed off host antibody which had attached to the surface of the tegument. It seems unlikely that this was due to breakage of the antigen-antibody linkages, but rather by the release of the entire immune complex consisting of host IgG linked to surface antigen. That the surface antigens were replaced by identical new moieties is shown by the fact that flukes which had rid their surface of a layer of IgG were immediately coated with a new layer DISCUSSION of adherent IgG when reexposed to immune During exposure to immune sheep serum serum. This control also served to show the juvenile flukes became completely that the progressive loss of ability to accept coated with IgG which adhered specifically fluorescent labeling was not due to deto the surface glycocalyx. The pattern of struction of Fc on the adherent host IgG. adhesion was identical whether the flukes Had that been the case the surface antigens were living or formalin fixed, and closely would have remained blocked and hence resembled that which occurred with fixed been unavailable for attachment of further Fusciola gigantica juveniles exposed to IgG on reexposure to antiserum. It is interesting to note that different immune bovine serum (Hanna and Jura 1977). The reticular appearance suggests areas of the fluke tegument replaced antithat the agglutination reaction occurred gens at different rates. The area just anmainly with antigenic determinants present terior to the ventral sucker seemed to be on those areas of the tegument between the most active in this respect, while replacespines. ment was slowest at the extreme anterior and posterior regions. Living flukes which were completely coated with adherent immunoglobulin lost Transmission electron microscopy of the this rapidly during incubation in a medium tegument of juvenile flukes revealed the without antiserum, but if the flukes were presence of flocculent material associated fixed before or immediately after exposure with the outer aspect of the apical memfrequent but the apical membrane retained its continuity (Fig. 10). As with flukes incubated for shorter periods, considerable quantities of flocculent material were attached at the exposed surface of the tegument. Those flukes incubated in medium containing serum but lacking cycloheximide underwent changes similar to those described above, but to a less marked extent. Fewer of the tegumental cells became totally depleted of TO bodies within 48 hr and a higher concentration of bodies was maintained at the surface syncytium.

FIG. 8. Fascida hepatica excysted in rirro. Surface syncytium (S) and tegumental cells of a fluke incubated for 24 hr in 10% immune sheep serum with 1 mA4 cycloheximide present. The tegumental cells, having lost most of their TO bodies, appear somewhat shrunken. TO bodies, which pass into the syncytium via connecting tubules (C) running between the muscle blocks, are seen discharging at the apical membrane (M). Large quantities of flocculent immune complex (F) are attached at the surface. Nuclei of the tegumental cells (N) and spines (Sp) are evident. x 15,750. FIG. 9. Fasciola hepatica excysted in vitro. Tegument of a fluke incubated for 48 hr in 10% immune sheep serum with 1 mA4cycloheximide present. TO bodies have disappeared from the cytoplasm of the tegumental cells (TC) which is seen as a thin layer around the nuclei (N). Few TO bodies remain in the surface syncytium (S). X 16,000. FIG. 10. Fusciola hepafica excysted in vitro. Surface syncytium (S) of a fluke incubated for 48 hr in 10% immune sheep serum with 1 mA4 cycloheximide present. Few TO bodies occur and the layer of cytoplasm is much thinner than in equivalent controls. The apical surface is relatively smooth, and membrane-bound portions of cytoplasm (B) bleb from it. x 16,000.

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brane of those flukes which were incubated in immune serum. This material probably represented the immune complex formed between host IgG and surface antigens. It was apparently quite loosely attached and frequently appeared to slough readily from the surface. It was undoubtedly analogous to the precipitate which formed when metacercariae were cultured in immune rat serum by Howell et al. (1977). This precipitate has since been shown to contain a functional antigen of the parasite capable of imparting significant protection in vaccinated rats against fluke challenge (Howell and Sandeman 1979). There was no evidence for sloughing of the surface membrane which was seen to possess the trilaminate structure typical of animal cells (Bennett and Threadgold 1973, 1975). Host IgG apparently complexed with antigenic determinants in the dense glycocalyx which was closely applied to the outer aspect of the apical membrance in untreated flukes, and which was continually lost and replaced. There was some evidence from the morphological studies that glycocalyx turnover occurred more rapidly in the presence of specific antiserum than in nonimmune serum. Thus surface replacement in juvenile Fasciofa hepatica resembled “membranocalyx” turnover in adult Schistosoma, although the composition and molecular organization of the deciduous material was clearly different. In Schistosoma the bounding membrane of multilaminate vesicles in the surface syncytium is believed to fuse with the surface plasmalemma releasing lamellate contents which spread over the tegument surface giving rise to the characteristic heptalaminate appearance (Wilson and Barnes 1977). The membranocalyx sloughs in culture medium (Kusel et al. 1975) and is rapidly replaced. Glycocalyx replacement in juvenile Fasciofa hepatica may be closely analogous to the loss of cercarial glycocalyx when young schistosomula of Schistosoma mansoni are

incubated with immune rat serum (Brink et al. 1977). It is not apparent from the present study whether detachment of the glycocalyx - IgG complex involved merely the shearing of the outer filamentous layer of oligosaccharide side chains from the underlying dense layer of the glycocalyx (Threadgold 1976) or whether the glycocalyx in its entirety was detached from the surface membrane. Further histochemical investigation is required to clarify this point. Maintenance of the glycocalyx was apparently achieved by exocrine secretion of the contents of TO bodies. Following extrusion of the contents it is likely that the limiting membrane of at least some TO bodies became incorporated in the surface membrane, gradually flattening into the general apical surface in response to elastic tension in the plasmalemma or internal pressure in the syncytium. As TO bodies discharged apically more moved up into the syncytium from the tegumental cells to replace the loss, so continual turnover of the glycocalyx was assured while supplies of TO bodies lasted. In normal circumstances it is likely that manufacture of TO bodies by the GEFUGolgi system in the tegumental cells can proceed sufficiently rapidly to ensure that IgG does not remain on the surface long enough to facilitate complement fixation and/or attachment of immunocompetent host cells. However, in the present experimental situation where protein synthesis was blocked by cycloheximide, TO bodies could not be replaced by the tegumental cells. Their numbers dwindled as incubation in immune serum proceeded until finally the stores in the tegumental cells were depleted and few remained in the surface syncytium. For immunological damage to proceed further, it is likely that eosinophil intervention would be required (Butterworth 1977; Davies and Goose 1979). Even in the absence of cycloheximide the numbers of TO bodies diminished faster

Fasciolo

hepatica:

GLYCOCALYX

with incubation in immune serum than in nonimmune serum. This suggests that under the in vitro conditions employed synthesis of TO bodies in the tegumental cells was not vigorous enough to replace losses at the surface. Bennett and Threadgold (1975) noted that the tegumental cells of juvenile flukes which had spent 12 hr in the abdominal cavity of a mouse contained fewer TO bodies than did those of newly excysted flukes. Subsequently, however, the tegumental cells recovered and continued to produce TO bodies from their Golgi complexes. During development the tegumental cells have been shown to switch to production of smaller dense spherical entities known as Tl bodies which are characteristic of the adult fluke tegument (Bennett and Threadgold 1975). It has been shown by electron microscope autoradiography that the Tl bodies, which originate in Type 1 tegumental cells (Threadgold 1963, 1967), do indeed discharge their contents at the apical plasma membrane in adult flukes (Hanna 1980) so it is likely that glycocalyx turnover continues throughout the life of the fluke, albeit at a slower rate in the immunologically “safe” environment of the bile ducts. The mechanism is likely to be most important early in development once specific immunoglobulin has built up to a significant level in the host’s bloodstream and the parasite is still in the abdominal cavity or liver parenchyma. The occurrence of large stores of TO bodies in the tegumental cells of the metacercaria may be a preadaption enabling the newly excysted juvenile to counteract immune attack in a previously sensitized host. Thus a primary infection of Fasciola hepatica in sheep does not prevent the development of flukes in a challenge infection. The situation in “unnatural” definitive hosts such as rats and cattle may be different since these animals develop substantial resistance to challenge with Fasciola hepatica following a primary

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infection (Smithers 1976). Perhaps the immunological response in these hosts is more vigorous than is the case in the “natural” host with which the fluke has had a long evolutionary relationship. The level of immune activity may be sufficiently high that it depletes the stores of TO bodies and outpaces production in the early stages of infection. Thus the fluke becomes susceptible to attack by complement and/or immunocompetent cells. In this respect it is interesting to note that infected rats gradually lose their resistance to incoming metacercariae as the primary fluke burden ages (Hughes et al. 1977). This possibly coincides with a falloff in the level of specific antibody in the circulation (Movsesijan and Jovanovic 1975; Hanna and Jura 1977). ACKNOWLEDGMENTS 1 wish to express my sincere thanks to the staff of the Parasitology Division, Veterinary Research Laboratories, Stormont, for provision and care of the experimental animals. I am also grateful to Mr. D. Reid for expert technical assistance. REFERENCES ARMOUR, J., AND DARGIE, J. D. 1974. Immunity to hepatica in the rat. Successful transfer of immunity by lymphoid cells and by serum. Experimental Parasitology 35, 381-388.

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