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Immunological damage to Hymenolepis diminuta following a challenge infection in C57 mice
InremationalJoumalforPareritology Vol. 17, No. 3,~. f’rinred in Great Britain.
795~803,1987.
0020-75 19/87 $3.00 + 0.00 t’e’ergomon Journals Ltd. Q 1987Ausrralian Societyfor hrasirology.
IMMUNOLOGICAL DAMAGE TO HYMENOLEPIS DIMINUTA FOLLOWING A CHALLENGE INFECTION IN C57 MICE and D. W. HALTON
M. D. MCCAIGUE Department
of Zoology,
The Queen’s University, (Received
Belfast BT7 lNN, Northern
Ireland,
U.K.
13 March 1986)
Abstract-McCAIcuE M. D. and HALTON D. W. 1987. Immunological damage to Hymenolepis diminuta following a challenge infection in C57 mice. International Journal for Parasitology 17: 795-803. Ultrastructural observations were made on the scpleces of Hymenofepis diminuta from lo-worm 2” infections in C57 mice to determine whether the immune stress causing reduced establishment, stunting of growth and early rejection was accompanied by significant damage to the worm fine structure. In almost all of the worms recovered there was extensive damage to tissues throughout the scolex, even in those examined 24 h postsecondary-infection (p-2’-i). Many of the muscle, tegumental and parenchyma cells were completely disrupted and dispersed, leaving only isolated strands of cytoplasm and cell debris. A number of excretory ducts were breached, and large and apparently empty spaces were evident throughout the scolex tissues. There was an increase in lipid accumulation in the tegumental syncytium, underlying cytons and in the suckers. Extensive vacuolation also occurred in the sucker tegument. Although some scoleces showed
certain regions to be apparently undamaged, close examination revealed a fine structure consistent with enhanced metabolic activity. The structural abnormalities observed are discussed in relation to host immunity. INDEX KEY WORDS: Hymenolepis diminuta; C57 mice; primary infection; destrobilation; immune response; tegument; ultrastructure; blebbing; vacuolation; transmission electron microscopy.
INTRODUCTION
secondary infection; lipid accumulation;
severe immunologically-induced changes in fine structure associated with such dramatic events as destrobilation and stunting of worm growth. Moreover, the ultrastructure of almost every organ system of normal adult H. diminutu from the natural rat host has been thoroughly investigated and described (Ubelaker, Allison & Specian, 1973; Lumsden, 1975; Lumsden & Specian, 1980) and transmission electron microscopy has been used extensively to study morphological damage to other intestinal helminths under immune stress (Ogilvie & Hockley, 1968; Lee, 1969, 1971; Harness, Smith& Bland, 1973; Love, Ogilvie & McLaren, 1976). The aims of the present investigation are two-fold: firstly, to describe for the first time any major differences between normal worm ultrastructure and 2” worm ultrastructure which could be due to the
MICE given a primary (1”) infection of H. diminuta respond by inducing destrobilation and/or rejection of the worms and, in a secondary (2”) infection, cause severe stunting of worm growth. These events are now known to be immunologically mediated (Hopkins, Subramanian & Stallard, 1972a, b; Befus, 1975), although the actual mechanism causing worm expulsion or the metabolic changes which lead to destrobilation and stunting remain unknown. Recently, a study on H. diminuta scoleces has shown that approaching and during the destrobilation/rejection period of a 5-worm lOinfection in C57 mice, many worms display adverse changes in their fine structure (McCaigue, Halton & Hopkins, 1986). These changes were never observed in the scoleces of H. diminuta from S-worm infections of the normal rat host or from immunosuppressed mice, suggesting they are a consequence of the host’s 1” immune response. Prior to this last report only one previous ultrastructural study has been recorded on immunological damage to adult H. diminuta during 1” infections (Befus & Threadgold, 1975). Moreover, there is currently a complete lack of information on ultrastructural events which might be occurring in the tiny stunted worms characteristic of 2” infections. This is surprising since one would almost certainly anticipate
acquired immune resistance induced by a lo (immunizing) infection in mice and, secondly, to compare these changes with those which have emerged from the few studies of 1” infections. It was hoped that a profile of the fine structural alterations accompanying the stunting of growth and early rejection of 2” worms would contribute a better understanding of the underlying immune mechanisms in the adult cestode infection.
795
796
M. D. MCCAIGU~ and D. W. HAI:ION
MATERIALS
AND METHODS
The Rice strain (originally from Rice University, Houston, Texas) of !I. diminufa was used. Cysticercoids were reared in Tr&lium conjuum at 26-28’6, and an inbred strain of C57 mice (Bantin &Kineman Ltd., Hull, U.K.) maintained in an isolated worm-free environment served as experimental hosts. On day 0, 5-worm I’ infections were administered to mice (6-S weeks old) as described by McCaigue ef al. (1986). Following 3 weeks post-primary infection (p- 1O-i) when all the 1” worms are naturally rejected (rejection of a 5-worm IO-i is complete by day 12) the mice were challenged with a 2*-i of 10 cysticercoids administered as above. Worms recovered from 10 cysticercoid infections in the normal rat host (Wistar strain) served as controls. Hosts were autopsied at 24 h intervals post infection (p.i.) and the worms were recovered, fixed and prepared for transmission electron microscopy as described by McCaigue e/ ul. (1986). RESULTS
From 24 h p-2”-i up to at least day 5 p-2”-i extensive and significant ultrastructural damage occurs in the scoleces of H. diminuta recovered from 2” infections in C57 mice. Compared with control worms, where the cells and organ systems of the scolex remain intact and in close apposition (Figs. 1,2), the vast majority of the worms recovered from days l-5 p-2”-i showed large areas of scolex tissue in which the muscle, tegumental and parenchyma cells were completely disrupted and dispersed. There was also evidence that after only 24 h p-2”-i (Fig. 3) and 48 h p-2”-i (Fig. 4) several excretory ducts were breached, and their contents leaked into the surrounding area. Some scoleces displayed extensive separation of the cells and muscle bands immediately beneath the surface tegument, leaving only isolated strands of cytoplasm and cell debris in this region (Fig. 5); others, except for the presence of a few lipid droplets, showed an apparently normal undisrupted appearance of the cells and tissues close to the surface tegument, but with severe disruption evident in deeper regions (Fig. 6). In all cases, the tissue disruption resulted in the formation of large and apparently empty spaces or cavities throughout the scoleces (Figs. 3-6). Morphologically, the suckers appeared to be undamaged, at least by days 1 and 2 p-2”% However, by day 4 p-2”-i large accumulations of lipid occupied the sucker tissues (Fig. 7) and extensive vacuolation along the base of the sucker distal tegument was evident (Fig. 8). Elsewhere in the damaged scoleces, but notably in the
tegumental cytons and the surface syncytium itself, there was marked deposition of lipid, particularly from day 3 p-2? (Figs. 9, 10).
In scoleces where surface regions were superficially undamaged (Fig. 6) closer examination often revealed that large areas of the surface syncytium contained mitochondria that were more numerous and larger than normal (Fig. 1 I), indicating an increased metabolic activity. These areas also displayed an apparent increase in the population of discoidal secretory bodies (Fig. 13). In this connection, the subjacent tegumental cytons showed ultrastructural evidence of extensive synthetic activity, in that they contained many prominent Golgi fields in close association with numerous strands of granular endoplasmic reticulum (GER) and mitochondtia (Fig. 14). In places, the distal cytoplasm of these regions of tegument showed “blebbing” or blistering
of the surface plasma membrane (Fig. 12). Some preliminary observations revealed that in mice there was a reduced establishment of 2” worms, when compared with a 1” infection, since worm recovery was always much lower during a 2” than a 10 infection. Moreover, challenge worms were rejected sooner and their growth more severely stunted, with worms never reaching more than 1-2 mm in length, even by day 4 or 5 p-2”-i. DISCUSSION
This report describes for the first time the scolex fine structure of H. diminuta runts recovered from immunized mice. It is clear that from as early as 24 h, profound, deleterious changes occur in the ultrastructure of the scolex tegument and associated tissues during a 2” infection in C57 mice, almost to the point of complete cellular obliteration in some cases. Since tapeworms are without a gut, the tegument serves a digestive and absorptive role, as well as being the principal protective organ system. Thus, any disruption in its function would almost certainly result in the death and/or expulsion of the parasite. Previous observations on host-parasite systems suggest that the ultrastructural damage to the digestive-absorptive surfaces of parasites is one of the earliest manifestations of host immunity in vivo (Ogilvie & Hockley, 1968; Lee, 1969, 1971; Befus & Threadgold, 1975; Siebert, Good & Simmons, 1978; McCaigue et al., 1986). The data presented here also lend support to these
FIG. I. Low power section through the scolex of normal H. diminufu showing the natural compact association between the distal tegument (TG), underlying cells and tissues and the suckers (S). TC, tegumental cytons; MU, muscle; P, parenchyma. FIG. 2. Section through the central region of the scolex of normal H. diminutu showing the close association between cells and tissues both inside and outside the rostellar capsule. RMU, muscular wall of rostellar capsule; RTC, rostellar tegumental cyton; RP, rostellar parenchyma cells; SP, scolex parenchyma cells. FIG. 3. Section through the scolex of H. diminuta from a C57 mouse (24 h p-2*-i). Note how even after this time disruption of the cells and tissues has occurred leaving large empty spaces (*). many cell fragments (CF) and ruptured excretory ducts (ED). RMU, muscular band of rostellar capsule; TC, remnants of scolex tegumental cytons; S, sucker.
Immune damage to Hymenolepis diminuta
M. D. MCCAIGUE and D. W. HALTON
798
.
Immune
damage
to Hymenolepisdiminuta
observations. The gross disfiguration observed is probably a direct consequence of the fact that during a 2” infection, damage to the surface tegument has caused impairment of many of its important regulatory functions, such as membrane transport and osmoregulation. It is reasonable to infer that the resulting fluid and ionic exchanges are responsible for the cellular disruption observed. This further implies that the large and apparently empty spaces are, in fact, fluid-filled. Furthermore, the vacuolation in the surface tegument of the suckers is likely to be due to a fluid-forced swelling of the basal invaginations. Befus & Threadgold (1975) also presented evidence of vacuolation in the basal regions of the surface tegument of immune-damaged areas of H. diminutu, and suggested that changes in tegument function leading to fluid accumulation in basal invaginations was responsible. The actual immune mechanism leading to such degeneration is, as yet, unknown, but one possibility is that the damage to H. diminuta may be caused by specific host antibody or antibody plus complement. It has already been demonstrated unequivocally that the H. diminutu scolex is highly antigenic (Elowni, 1982; Hopkins& Barr, 1982). Thus, antibody or immune complexes on binding to the surface plasma membrane could block, or severely restrict nutrient uptake by the membrane carriers, or the direct lytic action of antibody plus complement may cause direct damage to the tegument. This hypothesis is firmly supported by Befus (1977) who demonstrated, using immunofluorescence, that in vivo host immunoglobulins (&A, IgG and IgM) plus the C, component of complement become bound to the surface of H. diminutu in mice, and that an increase in the production of host immunoglobulins is coincident with the time of worm rejection from a 1” infection. He proposed that these immunoglobulins were specific antibodies bound to antigens and could function by inhibiting worm metabolism. It was later reported that the tegument, and specifically the glycocalyx, was the in vivo immunoglobulin binding site in H. diminutu from mice (Threadgold & Befus, 1977). Antibody plus complement has been implicated as the cause of ultrastructural damage similar to that presented here for Tueniu crassiceps metacestodes (Siebert etal., 1978) and for the death of
799
T. taeniaeformis oncospheres in immunized mice (Mitchell, Goding & Rickard, 1977; Mitchell, Rajasekaxiah & Rickard, 1980). Furthermore, Bland (unpublished, The Immune Response of Mice to the Tapeworm Hymenolepis diminuta. PhD thesis, University of Glasgow, 1976) found that transport of 14C-labelled L-methionine and sodium acetate was less in H. diminuta from mice than in H. diminuta of the same weight from immunosuppressed mice or from rats. He concluded that there was specific depression of methionine and sodium acetate transport by an immune mediator in mice acting on tegumental transport loci. Thus, it appears that challenge worms in immunized mice undergo severe immune stress from a strong host immune response directed against the surface tegument. It is not unreasonable, therefore, to expect that normal tegumental function, particularly membrane transport and osmoregulation, would become greatly diminished. Host mucus on the worm surface may also be detrimental, since a marked increase in mucus production from intestinal goblet cells has been demonstrated in immune mice challenged with H. diminutu (McCaigue & Halton, unpublished). These events would also account for the reduced establishment, earlier rejection and drastic stunting of worm growth in 2” infections. It is highly likely that in immune mice, antibodies, mucus or both are produced rapidly and bound quickly to the antigenic surface. Thus, rapid inactivation of the digestive and transport properties of the tegument would restrict nutrient uptake and hence growth from a very early stage. Under such unfavourable conditions it is likely that fewer worms would become established, while others would be quickly overcome and rejected. Nevertheless, some worms do manage to survive for a short time in this hostile environment, albeit as damaged runts. Some obviously damaged scoleces still show a few regions which appear structurally normal (Fig. 6), suggesting at least some degree of normal tegumental function. The increased metabolic and synthetic activity of these regions indicates that the worms probably have some ability to repair the damaged tegument, or replace those areas of surface membrane and glycocalyx bearing absorbed host molecules. In the surface syncytium the increased numbers of discoidal secretory bodies containing
FIG. 4. Section through a central region of the scolex of H. diminuta from a C57 mouse devastation of the tissues by this time with vast open spaces (*), cell debris (CD) and ruptured RMU, muscular wall of rostellar capsule; S, sucker.
48 h p-r-i showing extensive excretory ducts (ED) ensuing.
FIG. 5. Section through the scolex of H. diminuta from a C57 mouse (day 4 p-2*-i) showing almost complete obliteration of the cells and muscle (MU) beneath the surface tegument (TG). Barely recognisable cell fragments (CF) and debris (D) are all that remain in the resulting huge spaces (*). S, sucker. FIG. 6. Section through the scolex of H. diminuta from a C57 mouse (day 5 p-2”-i). Although several lipid droplets (L) are present, the distal tegument (TG), muscle (MU) and neighbouring cells show the normal close apposition. However, in deeper regions large empty spaces (*) again result from the total fragmentation of the tissues. TC, tegumental cytons; P, parenchyma; S, sucker.
M. D. MCCA~WE and
FIG.,7.
Section through the sucker of H.
ofN.
D. IV. EIALIQN
rliminuru from a C57 mouse (day MU, muscle; P, par~nchyma.
4 p- 2*-i) showing dramatic lipid depositio
‘n
(L).
climirrura from a C57 mouse (day 4 p-2”-Qshowingextensivevacuolation (V) along hase of the sucker distal tegument (TC). MU, muscle. FIG. 9, Scolex distal cytoplasm of H. ~~rn~~~u~afrom a C57 mouse (day 3 p- Y-i). Note the presence of several large? lipid droplets (L) within the syncytium. DSB, discoidni secretory bodies. F?G. 10. Scolex tegumental cytons i,TC) of ii. ~~?~~~~~~from a C57 mouse (day 3 p-z”-ij d~s~l~yi~g extensive lipid acei imufation (L). Note also the lipid droplet in the distal tgument (TG). CC, cytoptasmic connection to surface sync) itium; N, nucleus; RM, basal plasma membrane; MU, muscle; DSB, discoidal secretory bodies. FIG..8.
Section through the sucker
the
Immune damage to Hymenolepis dimirtuta
801
FIG. 11. Seolex tegumentaI syncytium from an apparently undamaged region of the scolex of H. d~~i~u~ufrom a C57 mouse (day 4 p-2’if. However, note the numerous large mitochondria (M) indicative of a meta~I~calIy very active surface.BM, basal membrane; BI, basal invagination; MU, muscle. FIG. 12. Scolex tegumental syncytium from an apparently undamaged region of the scolex of H. diminutufrom a C57 mouse (day 4 p-2’i). However, note the bleb-like swehing (3) of the apical plasma membrane (PM). FIG. 13. Section through the scolex te~ment~ syncyti~m of H. d~~~~utu from a C57 mouse (day 5 p-Y-i) showing dense populations of discoidal secretory bodies (arrows). FIG. 14. Scolex tegumentaI cytons from an apparently undamaged region of the scolex of H. diminuta from a C57 mouse (day 4 p-y-i). Clearly the cytons are syntheticgly very active as evidenced by the numerous large Go&i fields (G), associated granular endoplasmic reticulum (GER) and mitochondria @I).
802
M. D. MCCAIGUE and D. W. HALTON
glycoprotein for renewal of the surface glycocalyx (Oaks & Lumsden, 197 1) supports this view. Also the subtegumental cytons have a cytoplasm containing a more than normal abundance of GER and Golgi bodies, features typical of cells that are synthesizing protein (or glycoprotein) for export from the cell. Thus, there appears to be a marked response by the worms to counter the host’s immune attack by increasing surface turnover and thereby maintaining a functional surface plasma membrane and associated transport systems. However, the swelling and blistering of the tegument in these regions suggest impending breakdown in the continuity and integrity of the worms’ surface, as a prelude to them finally succumbing to the host immune response and being rejected. Similar tegumental blebs are typical of H. diminuta and other cestodes when exposed to other forms of stress such as drugs (Becker, Melhorn, Andrews & Thomas, 198 1). Lipid accumulation may be the result of its initial rapid uptake, stimulated to supply membrane precursors for increased production of membrane-bound secretory bodies and for surface turnover. Since lipid excretion is an energy-required process, it is likely to be inhibited in worms under stress. In this way, excess or unusable lipid would accumulate, accounting for the large deposits found in the present study. It is also known that lipid accumulation is characteristic of degenerative changes associated with cell injury (Dixon, 1970), and has been recorded for immunologically damaged Nippostron&ts brasiliensis (by Lee, 1969, 1971). Several of the events described here are remarkably similar to some reported previously during the destrobilation/rejection of a lOinfection in mice (McCaigue et al., 1986). This, together with the reduced establishment and earlier 2’worm rejection, indicate that a similar, but much more rapid and intense response is operating in the challenge infection. It also highlights a fundamental characteristic of 2”immunity, namely that the strong resistance to challenge worms is the result of memory from the immunizing 1” infection. Acknowledgemenfs-The work was undertaken while M. D. McC. was in receipt of a Postgraduate Studentship from the Department of Education (NJ.).
REFERENCES BECKER B., MELHORN H., ANDREWS P. &THOMAS H. 1981. Ultrastructural investigations on the effect of praziquantel on the tegument of five species of cestodes. Zeitschriftfiir Parasitenkunde 64: 257-269. BEFUS A. D. 1975. Secondary infections of Hymenolepis diminuta in mice: effects of varying worm burdens in primary and secondary infections. Parasitology 7 1: 6 I75. BEFUS A. D. 1977. Hymenolepis diminuta and H. microstoma : mouse immunoglobulins binding to the tegumental surface. Experimental Parasitology 4 1: 242-25 1.
Bs~us A. D. & THKEADFOLD L. 7‘. 1975. Possible immunological damage to the tegument of Hymmolepis diminuttr in mice and rats. Parasifology 71: 525-534. DIXON K. C. 1970. Disorders of the cell. In: Companion to MedicalStudies (Edited byPAssMoRE R. & ROBSON .I. S.), Vol. 2, pp. 25.1-25.44. Blackwell Scientific Publications, Oxford. ELOWNI E. E. 19X2. Hymenolepis diminuta: The origin of protective antigens Experimental Parasitology 53: 157163. HARNESS E., SMITH K. & BLAND P. 1973. Structural changes in the bovine nematode Haemonchusplacei, that may be associated with host immune response. Parusitology 66: 199-205. HOPKINS C. A. & BARR 1. F. 1982. The source of antigen in an adult tapeworm. Internationalfournalfor Parasitology 12: 327-333. HOPKINS C. A., SUBRAMANIANG. & STALLARDH. E. 1972a. The development of Hymenolepis diminuta in primary and secondary infections in mice. Parasitology 64: 40 l412. HOPKINS, C. A., SUBKAMANIANG. & STALLARD H. E. 1972b. The effect of immunosuppressants on the development of Hymenolepis diminuta in mice. Parasitolo& 65: 11 l- 1I&. LEE D. L. 1969. Changes in adult Nioaostronavlus brasiliensis during the development of immunity to this nematode in rats. 1. Changes in ultrastructure. Parasitology 59: 29-39. LEE D. L. 197 1. Changes in adult Nipposfrongylus brasiliensis during the development of immunity to this nematode in rats. 2. Total lipids and neutral lipids. Parasitology 63: 271-274. LOVE R. J., OGILVIE B. M. & MCLAREN D. J. 1976. The immune mechanism which expels the intestinal stage of Trichinella spiralis from rats. Immunology 30: 7-15. LUMSDEN R. D. 1975. Surface ultrastructure and cytochemistry of parasitic helminths. Experimental Parasitology 37: 267-339. LUMSDEN R. D. & SPECIAN R. 1980. The morphology, histology and fine structure of the adult stage of the cyclophyllidean tapeworm Hymenolepis diminuta. In: Biology of the Tapeworm Hymenolepis diminuta (Edited by ARAI H. P.), pp. 157-240. Academic Press, New York. MCCAIGUE M. D., HALTON D. W. & HOPKINS C. A. 1986. Hymenolepis diminuta ultrastructural abnormalities in worms from C57 mice. Experimental Parasitology 62: 51-50. MITCHELL G. F., GODING J. W. & RICKARD M. D. 1977. Studies on immune responses to larval cestodes in mice. Increased susceptibility of certain mouse strains and hypothymic mice to Taenia taeniaeformis and analysis of passive transfer of resistance with serum. Australian Journal of Experimental Biology and Medical Science 58: 82-103. MITCHELL G. F., RAJASEKARIAH G. R. & RICKARD M. D. 1980. A mechanism to account for mouse strain variation in resistance to the larval cestode, Taenia taeniaeformis. Immunology 39: 481-489. OAKS J. A. & LUMSDEN R. D. 197 1. Cytological studies on the absorptive surfaces of cestodes. V. Incorporation of carbohydiate-containing macromolecules in& tegument membranes. JournalofParasitolonv 57: 1256-1268. OGILVIE B. M. & HOCKS D. J. 1968. Effects of immunity on Nippostrongylus brasiliensis adult worms: reversible and irreversible changes in infectivity, reproduction and morphology. Journal of Parasitology 54: 1073-l 048. Y
1
I
-,
Immune SIEBERT
damage
to Hymenolepis diminuta
A. E., GOOD A. H. & SIMMONS J. E. 1978. Ultrastructural aspects of early immune damage to Taenia crassiceps metacestodes. International Journalfor Parasitology 8: 45-53. THREADGOLD L. T. & BEFUS A. D. 1977. Hymenolepis diminura: Ultrastructural localization of immunoglobulin-
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binding sites on the tegument. Experimental Parasitology 43: 169-179. UBELAKER J., ALLISON V. & SPECIAN R. 1973. Surface topography of Hymenolepis diminula by scanning electron microscopy. Journal of Parasitology 59: 667-67 1.
795~803,1987.
0020-75 19/87 $3.00 + 0.00 t’e’ergomon Journals Ltd. Q 1987Ausrralian Societyfor hrasirology.
IMMUNOLOGICAL DAMAGE TO HYMENOLEPIS DIMINUTA FOLLOWING A CHALLENGE INFECTION IN C57 MICE and D. W. HALTON
M. D. MCCAIGUE Department
of Zoology,
The Queen’s University, (Received
Belfast BT7 lNN, Northern
Ireland,
U.K.
13 March 1986)
Abstract-McCAIcuE M. D. and HALTON D. W. 1987. Immunological damage to Hymenolepis diminuta following a challenge infection in C57 mice. International Journal for Parasitology 17: 795-803. Ultrastructural observations were made on the scpleces of Hymenofepis diminuta from lo-worm 2” infections in C57 mice to determine whether the immune stress causing reduced establishment, stunting of growth and early rejection was accompanied by significant damage to the worm fine structure. In almost all of the worms recovered there was extensive damage to tissues throughout the scolex, even in those examined 24 h postsecondary-infection (p-2’-i). Many of the muscle, tegumental and parenchyma cells were completely disrupted and dispersed, leaving only isolated strands of cytoplasm and cell debris. A number of excretory ducts were breached, and large and apparently empty spaces were evident throughout the scolex tissues. There was an increase in lipid accumulation in the tegumental syncytium, underlying cytons and in the suckers. Extensive vacuolation also occurred in the sucker tegument. Although some scoleces showed
certain regions to be apparently undamaged, close examination revealed a fine structure consistent with enhanced metabolic activity. The structural abnormalities observed are discussed in relation to host immunity. INDEX KEY WORDS: Hymenolepis diminuta; C57 mice; primary infection; destrobilation; immune response; tegument; ultrastructure; blebbing; vacuolation; transmission electron microscopy.
INTRODUCTION
secondary infection; lipid accumulation;
severe immunologically-induced changes in fine structure associated with such dramatic events as destrobilation and stunting of worm growth. Moreover, the ultrastructure of almost every organ system of normal adult H. diminutu from the natural rat host has been thoroughly investigated and described (Ubelaker, Allison & Specian, 1973; Lumsden, 1975; Lumsden & Specian, 1980) and transmission electron microscopy has been used extensively to study morphological damage to other intestinal helminths under immune stress (Ogilvie & Hockley, 1968; Lee, 1969, 1971; Harness, Smith& Bland, 1973; Love, Ogilvie & McLaren, 1976). The aims of the present investigation are two-fold: firstly, to describe for the first time any major differences between normal worm ultrastructure and 2” worm ultrastructure which could be due to the
MICE given a primary (1”) infection of H. diminuta respond by inducing destrobilation and/or rejection of the worms and, in a secondary (2”) infection, cause severe stunting of worm growth. These events are now known to be immunologically mediated (Hopkins, Subramanian & Stallard, 1972a, b; Befus, 1975), although the actual mechanism causing worm expulsion or the metabolic changes which lead to destrobilation and stunting remain unknown. Recently, a study on H. diminuta scoleces has shown that approaching and during the destrobilation/rejection period of a 5-worm lOinfection in C57 mice, many worms display adverse changes in their fine structure (McCaigue, Halton & Hopkins, 1986). These changes were never observed in the scoleces of H. diminuta from S-worm infections of the normal rat host or from immunosuppressed mice, suggesting they are a consequence of the host’s 1” immune response. Prior to this last report only one previous ultrastructural study has been recorded on immunological damage to adult H. diminuta during 1” infections (Befus & Threadgold, 1975). Moreover, there is currently a complete lack of information on ultrastructural events which might be occurring in the tiny stunted worms characteristic of 2” infections. This is surprising since one would almost certainly anticipate
acquired immune resistance induced by a lo (immunizing) infection in mice and, secondly, to compare these changes with those which have emerged from the few studies of 1” infections. It was hoped that a profile of the fine structural alterations accompanying the stunting of growth and early rejection of 2” worms would contribute a better understanding of the underlying immune mechanisms in the adult cestode infection.
795
796
M. D. MCCAIGU~ and D. W. HAI:ION
MATERIALS
AND METHODS
The Rice strain (originally from Rice University, Houston, Texas) of !I. diminufa was used. Cysticercoids were reared in Tr&lium conjuum at 26-28’6, and an inbred strain of C57 mice (Bantin &Kineman Ltd., Hull, U.K.) maintained in an isolated worm-free environment served as experimental hosts. On day 0, 5-worm I’ infections were administered to mice (6-S weeks old) as described by McCaigue ef al. (1986). Following 3 weeks post-primary infection (p- 1O-i) when all the 1” worms are naturally rejected (rejection of a 5-worm IO-i is complete by day 12) the mice were challenged with a 2*-i of 10 cysticercoids administered as above. Worms recovered from 10 cysticercoid infections in the normal rat host (Wistar strain) served as controls. Hosts were autopsied at 24 h intervals post infection (p.i.) and the worms were recovered, fixed and prepared for transmission electron microscopy as described by McCaigue e/ ul. (1986). RESULTS
From 24 h p-2”-i up to at least day 5 p-2”-i extensive and significant ultrastructural damage occurs in the scoleces of H. diminuta recovered from 2” infections in C57 mice. Compared with control worms, where the cells and organ systems of the scolex remain intact and in close apposition (Figs. 1,2), the vast majority of the worms recovered from days l-5 p-2”-i showed large areas of scolex tissue in which the muscle, tegumental and parenchyma cells were completely disrupted and dispersed. There was also evidence that after only 24 h p-2”-i (Fig. 3) and 48 h p-2”-i (Fig. 4) several excretory ducts were breached, and their contents leaked into the surrounding area. Some scoleces displayed extensive separation of the cells and muscle bands immediately beneath the surface tegument, leaving only isolated strands of cytoplasm and cell debris in this region (Fig. 5); others, except for the presence of a few lipid droplets, showed an apparently normal undisrupted appearance of the cells and tissues close to the surface tegument, but with severe disruption evident in deeper regions (Fig. 6). In all cases, the tissue disruption resulted in the formation of large and apparently empty spaces or cavities throughout the scoleces (Figs. 3-6). Morphologically, the suckers appeared to be undamaged, at least by days 1 and 2 p-2”% However, by day 4 p-2”-i large accumulations of lipid occupied the sucker tissues (Fig. 7) and extensive vacuolation along the base of the sucker distal tegument was evident (Fig. 8). Elsewhere in the damaged scoleces, but notably in the
tegumental cytons and the surface syncytium itself, there was marked deposition of lipid, particularly from day 3 p-2? (Figs. 9, 10).
In scoleces where surface regions were superficially undamaged (Fig. 6) closer examination often revealed that large areas of the surface syncytium contained mitochondria that were more numerous and larger than normal (Fig. 1 I), indicating an increased metabolic activity. These areas also displayed an apparent increase in the population of discoidal secretory bodies (Fig. 13). In this connection, the subjacent tegumental cytons showed ultrastructural evidence of extensive synthetic activity, in that they contained many prominent Golgi fields in close association with numerous strands of granular endoplasmic reticulum (GER) and mitochondtia (Fig. 14). In places, the distal cytoplasm of these regions of tegument showed “blebbing” or blistering
of the surface plasma membrane (Fig. 12). Some preliminary observations revealed that in mice there was a reduced establishment of 2” worms, when compared with a 1” infection, since worm recovery was always much lower during a 2” than a 10 infection. Moreover, challenge worms were rejected sooner and their growth more severely stunted, with worms never reaching more than 1-2 mm in length, even by day 4 or 5 p-2”-i. DISCUSSION
This report describes for the first time the scolex fine structure of H. diminuta runts recovered from immunized mice. It is clear that from as early as 24 h, profound, deleterious changes occur in the ultrastructure of the scolex tegument and associated tissues during a 2” infection in C57 mice, almost to the point of complete cellular obliteration in some cases. Since tapeworms are without a gut, the tegument serves a digestive and absorptive role, as well as being the principal protective organ system. Thus, any disruption in its function would almost certainly result in the death and/or expulsion of the parasite. Previous observations on host-parasite systems suggest that the ultrastructural damage to the digestive-absorptive surfaces of parasites is one of the earliest manifestations of host immunity in vivo (Ogilvie & Hockley, 1968; Lee, 1969, 1971; Befus & Threadgold, 1975; Siebert, Good & Simmons, 1978; McCaigue et al., 1986). The data presented here also lend support to these
FIG. I. Low power section through the scolex of normal H. diminufu showing the natural compact association between the distal tegument (TG), underlying cells and tissues and the suckers (S). TC, tegumental cytons; MU, muscle; P, parenchyma. FIG. 2. Section through the central region of the scolex of normal H. diminutu showing the close association between cells and tissues both inside and outside the rostellar capsule. RMU, muscular wall of rostellar capsule; RTC, rostellar tegumental cyton; RP, rostellar parenchyma cells; SP, scolex parenchyma cells. FIG. 3. Section through the scolex of H. diminuta from a C57 mouse (24 h p-2*-i). Note how even after this time disruption of the cells and tissues has occurred leaving large empty spaces (*). many cell fragments (CF) and ruptured excretory ducts (ED). RMU, muscular band of rostellar capsule; TC, remnants of scolex tegumental cytons; S, sucker.
Immune damage to Hymenolepis diminuta
M. D. MCCAIGUE and D. W. HALTON
798
.
Immune
damage
to Hymenolepisdiminuta
observations. The gross disfiguration observed is probably a direct consequence of the fact that during a 2” infection, damage to the surface tegument has caused impairment of many of its important regulatory functions, such as membrane transport and osmoregulation. It is reasonable to infer that the resulting fluid and ionic exchanges are responsible for the cellular disruption observed. This further implies that the large and apparently empty spaces are, in fact, fluid-filled. Furthermore, the vacuolation in the surface tegument of the suckers is likely to be due to a fluid-forced swelling of the basal invaginations. Befus & Threadgold (1975) also presented evidence of vacuolation in the basal regions of the surface tegument of immune-damaged areas of H. diminutu, and suggested that changes in tegument function leading to fluid accumulation in basal invaginations was responsible. The actual immune mechanism leading to such degeneration is, as yet, unknown, but one possibility is that the damage to H. diminuta may be caused by specific host antibody or antibody plus complement. It has already been demonstrated unequivocally that the H. diminutu scolex is highly antigenic (Elowni, 1982; Hopkins& Barr, 1982). Thus, antibody or immune complexes on binding to the surface plasma membrane could block, or severely restrict nutrient uptake by the membrane carriers, or the direct lytic action of antibody plus complement may cause direct damage to the tegument. This hypothesis is firmly supported by Befus (1977) who demonstrated, using immunofluorescence, that in vivo host immunoglobulins (&A, IgG and IgM) plus the C, component of complement become bound to the surface of H. diminutu in mice, and that an increase in the production of host immunoglobulins is coincident with the time of worm rejection from a 1” infection. He proposed that these immunoglobulins were specific antibodies bound to antigens and could function by inhibiting worm metabolism. It was later reported that the tegument, and specifically the glycocalyx, was the in vivo immunoglobulin binding site in H. diminutu from mice (Threadgold & Befus, 1977). Antibody plus complement has been implicated as the cause of ultrastructural damage similar to that presented here for Tueniu crassiceps metacestodes (Siebert etal., 1978) and for the death of
799
T. taeniaeformis oncospheres in immunized mice (Mitchell, Goding & Rickard, 1977; Mitchell, Rajasekaxiah & Rickard, 1980). Furthermore, Bland (unpublished, The Immune Response of Mice to the Tapeworm Hymenolepis diminuta. PhD thesis, University of Glasgow, 1976) found that transport of 14C-labelled L-methionine and sodium acetate was less in H. diminuta from mice than in H. diminuta of the same weight from immunosuppressed mice or from rats. He concluded that there was specific depression of methionine and sodium acetate transport by an immune mediator in mice acting on tegumental transport loci. Thus, it appears that challenge worms in immunized mice undergo severe immune stress from a strong host immune response directed against the surface tegument. It is not unreasonable, therefore, to expect that normal tegumental function, particularly membrane transport and osmoregulation, would become greatly diminished. Host mucus on the worm surface may also be detrimental, since a marked increase in mucus production from intestinal goblet cells has been demonstrated in immune mice challenged with H. diminutu (McCaigue & Halton, unpublished). These events would also account for the reduced establishment, earlier rejection and drastic stunting of worm growth in 2” infections. It is highly likely that in immune mice, antibodies, mucus or both are produced rapidly and bound quickly to the antigenic surface. Thus, rapid inactivation of the digestive and transport properties of the tegument would restrict nutrient uptake and hence growth from a very early stage. Under such unfavourable conditions it is likely that fewer worms would become established, while others would be quickly overcome and rejected. Nevertheless, some worms do manage to survive for a short time in this hostile environment, albeit as damaged runts. Some obviously damaged scoleces still show a few regions which appear structurally normal (Fig. 6), suggesting at least some degree of normal tegumental function. The increased metabolic and synthetic activity of these regions indicates that the worms probably have some ability to repair the damaged tegument, or replace those areas of surface membrane and glycocalyx bearing absorbed host molecules. In the surface syncytium the increased numbers of discoidal secretory bodies containing
FIG. 4. Section through a central region of the scolex of H. diminuta from a C57 mouse devastation of the tissues by this time with vast open spaces (*), cell debris (CD) and ruptured RMU, muscular wall of rostellar capsule; S, sucker.
48 h p-r-i showing extensive excretory ducts (ED) ensuing.
FIG. 5. Section through the scolex of H. diminuta from a C57 mouse (day 4 p-2*-i) showing almost complete obliteration of the cells and muscle (MU) beneath the surface tegument (TG). Barely recognisable cell fragments (CF) and debris (D) are all that remain in the resulting huge spaces (*). S, sucker. FIG. 6. Section through the scolex of H. diminuta from a C57 mouse (day 5 p-2”-i). Although several lipid droplets (L) are present, the distal tegument (TG), muscle (MU) and neighbouring cells show the normal close apposition. However, in deeper regions large empty spaces (*) again result from the total fragmentation of the tissues. TC, tegumental cytons; P, parenchyma; S, sucker.
M. D. MCCA~WE and
FIG.,7.
Section through the sucker of H.
ofN.
D. IV. EIALIQN
rliminuru from a C57 mouse (day MU, muscle; P, par~nchyma.
4 p- 2*-i) showing dramatic lipid depositio
‘n
(L).
climirrura from a C57 mouse (day 4 p-2”-Qshowingextensivevacuolation (V) along hase of the sucker distal tegument (TC). MU, muscle. FIG. 9, Scolex distal cytoplasm of H. ~~rn~~~u~afrom a C57 mouse (day 3 p- Y-i). Note the presence of several large? lipid droplets (L) within the syncytium. DSB, discoidni secretory bodies. F?G. 10. Scolex tegumental cytons i,TC) of ii. ~~?~~~~~~from a C57 mouse (day 3 p-z”-ij d~s~l~yi~g extensive lipid acei imufation (L). Note also the lipid droplet in the distal tgument (TG). CC, cytoptasmic connection to surface sync) itium; N, nucleus; RM, basal plasma membrane; MU, muscle; DSB, discoidal secretory bodies. FIG..8.
Section through the sucker
the
Immune damage to Hymenolepis dimirtuta
801
FIG. 11. Seolex tegumentaI syncytium from an apparently undamaged region of the scolex of H. d~~i~u~ufrom a C57 mouse (day 4 p-2’if. However, note the numerous large mitochondria (M) indicative of a meta~I~calIy very active surface.BM, basal membrane; BI, basal invagination; MU, muscle. FIG. 12. Scolex tegumental syncytium from an apparently undamaged region of the scolex of H. diminutufrom a C57 mouse (day 4 p-2’i). However, note the bleb-like swehing (3) of the apical plasma membrane (PM). FIG. 13. Section through the scolex te~ment~ syncyti~m of H. d~~~~utu from a C57 mouse (day 5 p-Y-i) showing dense populations of discoidal secretory bodies (arrows). FIG. 14. Scolex tegumentaI cytons from an apparently undamaged region of the scolex of H. diminuta from a C57 mouse (day 4 p-y-i). Clearly the cytons are syntheticgly very active as evidenced by the numerous large Go&i fields (G), associated granular endoplasmic reticulum (GER) and mitochondria @I).
802
M. D. MCCAIGUE and D. W. HALTON
glycoprotein for renewal of the surface glycocalyx (Oaks & Lumsden, 197 1) supports this view. Also the subtegumental cytons have a cytoplasm containing a more than normal abundance of GER and Golgi bodies, features typical of cells that are synthesizing protein (or glycoprotein) for export from the cell. Thus, there appears to be a marked response by the worms to counter the host’s immune attack by increasing surface turnover and thereby maintaining a functional surface plasma membrane and associated transport systems. However, the swelling and blistering of the tegument in these regions suggest impending breakdown in the continuity and integrity of the worms’ surface, as a prelude to them finally succumbing to the host immune response and being rejected. Similar tegumental blebs are typical of H. diminuta and other cestodes when exposed to other forms of stress such as drugs (Becker, Melhorn, Andrews & Thomas, 198 1). Lipid accumulation may be the result of its initial rapid uptake, stimulated to supply membrane precursors for increased production of membrane-bound secretory bodies and for surface turnover. Since lipid excretion is an energy-required process, it is likely to be inhibited in worms under stress. In this way, excess or unusable lipid would accumulate, accounting for the large deposits found in the present study. It is also known that lipid accumulation is characteristic of degenerative changes associated with cell injury (Dixon, 1970), and has been recorded for immunologically damaged Nippostron&ts brasiliensis (by Lee, 1969, 1971). Several of the events described here are remarkably similar to some reported previously during the destrobilation/rejection of a lOinfection in mice (McCaigue et al., 1986). This, together with the reduced establishment and earlier 2’worm rejection, indicate that a similar, but much more rapid and intense response is operating in the challenge infection. It also highlights a fundamental characteristic of 2”immunity, namely that the strong resistance to challenge worms is the result of memory from the immunizing 1” infection. Acknowledgemenfs-The work was undertaken while M. D. McC. was in receipt of a Postgraduate Studentship from the Department of Education (NJ.).
REFERENCES BECKER B., MELHORN H., ANDREWS P. &THOMAS H. 1981. Ultrastructural investigations on the effect of praziquantel on the tegument of five species of cestodes. Zeitschriftfiir Parasitenkunde 64: 257-269. BEFUS A. D. 1975. Secondary infections of Hymenolepis diminuta in mice: effects of varying worm burdens in primary and secondary infections. Parasitology 7 1: 6 I75. BEFUS A. D. 1977. Hymenolepis diminuta and H. microstoma : mouse immunoglobulins binding to the tegumental surface. Experimental Parasitology 4 1: 242-25 1.
Bs~us A. D. & THKEADFOLD L. 7‘. 1975. Possible immunological damage to the tegument of Hymmolepis diminuttr in mice and rats. Parasifology 71: 525-534. DIXON K. C. 1970. Disorders of the cell. In: Companion to MedicalStudies (Edited byPAssMoRE R. & ROBSON .I. S.), Vol. 2, pp. 25.1-25.44. Blackwell Scientific Publications, Oxford. ELOWNI E. E. 19X2. Hymenolepis diminuta: The origin of protective antigens Experimental Parasitology 53: 157163. HARNESS E., SMITH K. & BLAND P. 1973. Structural changes in the bovine nematode Haemonchusplacei, that may be associated with host immune response. Parusitology 66: 199-205. HOPKINS C. A. & BARR 1. F. 1982. The source of antigen in an adult tapeworm. Internationalfournalfor Parasitology 12: 327-333. HOPKINS C. A., SUBRAMANIANG. & STALLARDH. E. 1972a. The development of Hymenolepis diminuta in primary and secondary infections in mice. Parasitology 64: 40 l412. HOPKINS, C. A., SUBKAMANIANG. & STALLARD H. E. 1972b. The effect of immunosuppressants on the development of Hymenolepis diminuta in mice. Parasitolo& 65: 11 l- 1I&. LEE D. L. 1969. Changes in adult Nioaostronavlus brasiliensis during the development of immunity to this nematode in rats. 1. Changes in ultrastructure. Parasitology 59: 29-39. LEE D. L. 197 1. Changes in adult Nipposfrongylus brasiliensis during the development of immunity to this nematode in rats. 2. Total lipids and neutral lipids. Parasitology 63: 271-274. LOVE R. J., OGILVIE B. M. & MCLAREN D. J. 1976. The immune mechanism which expels the intestinal stage of Trichinella spiralis from rats. Immunology 30: 7-15. LUMSDEN R. D. 1975. Surface ultrastructure and cytochemistry of parasitic helminths. Experimental Parasitology 37: 267-339. LUMSDEN R. D. & SPECIAN R. 1980. The morphology, histology and fine structure of the adult stage of the cyclophyllidean tapeworm Hymenolepis diminuta. In: Biology of the Tapeworm Hymenolepis diminuta (Edited by ARAI H. P.), pp. 157-240. Academic Press, New York. MCCAIGUE M. D., HALTON D. W. & HOPKINS C. A. 1986. Hymenolepis diminuta ultrastructural abnormalities in worms from C57 mice. Experimental Parasitology 62: 51-50. MITCHELL G. F., GODING J. W. & RICKARD M. D. 1977. Studies on immune responses to larval cestodes in mice. Increased susceptibility of certain mouse strains and hypothymic mice to Taenia taeniaeformis and analysis of passive transfer of resistance with serum. Australian Journal of Experimental Biology and Medical Science 58: 82-103. MITCHELL G. F., RAJASEKARIAH G. R. & RICKARD M. D. 1980. A mechanism to account for mouse strain variation in resistance to the larval cestode, Taenia taeniaeformis. Immunology 39: 481-489. OAKS J. A. & LUMSDEN R. D. 197 1. Cytological studies on the absorptive surfaces of cestodes. V. Incorporation of carbohydiate-containing macromolecules in& tegument membranes. JournalofParasitolonv 57: 1256-1268. OGILVIE B. M. & HOCKS D. J. 1968. Effects of immunity on Nippostrongylus brasiliensis adult worms: reversible and irreversible changes in infectivity, reproduction and morphology. Journal of Parasitology 54: 1073-l 048. Y
1
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to Hymenolepis diminuta
A. E., GOOD A. H. & SIMMONS J. E. 1978. Ultrastructural aspects of early immune damage to Taenia crassiceps metacestodes. International Journalfor Parasitology 8: 45-53. THREADGOLD L. T. & BEFUS A. D. 1977. Hymenolepis diminura: Ultrastructural localization of immunoglobulin-
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binding sites on the tegument. Experimental Parasitology 43: 169-179. UBELAKER J., ALLISON V. & SPECIAN R. 1973. Surface topography of Hymenolepis diminula by scanning electron microscopy. Journal of Parasitology 59: 667-67 1.