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The mode of passive protection against Hymenolepis nana induced by serum transfer
Inlernational
Journal
for
Parasitology.
1977.
Vol. 7. pp. 67-71.
Pergamon
Press. Printed
in Great
Britain.
THE MODE OF PASSIVE PROTECTION AGAINST ~Y~~~Q~~PI~ NANA INDUCED BY SERUM TRANSFER A. IT0 Department
of Medical Biology, School of Medicine, Showa University, Hatanodai, Shinagawaku, Tokyo, Japan (Received 23 February 1976; in revised form 6 July 1976)
A. 1977. The mode of passive protection against Hymenu~ep~s nana induced by serum transfer. international ~uur~~ for Paru~~to~~gy 7: 67-71. A protective immunity against the cestode ~ymeno~epis nana was transferred with serum taken from actively immunized mice. All of 17 pooled sera examined, which were taken from mice immunized for 3 or more weeks, were strongly effective. Intraperitoneal injection of a total of 3.0 ml serum made the recipient mice (44 weeks old) almost completely immune. In almost all the mice given immune serum no cysticercoids were found on day 4. In mice receiving immune serum, oncospheres hatched, invaded the intestinal villi and differentiated to stage II or III larvae, but failed to develop to fully developed cysticercoids. The degree of protection conferred by serum transfer was similar to, but slightly weaker than that stimulated by active immuni~tion. The major effect of immune serum was damaging hatched oncospheres in both the intestinal lumen and the villi within 1 day post infection. Abstract-ITo
INDEX KEY WORDS: oncosphere; cysticercoid.
Hymenolepis
nana;
mice; intestine;
immunity;
serum transfer;
immunity against H. nann reinfection is thymus dependent (Okamoto, 1968; Okamoto & Koizumi, 1972). However, it is well-known that the antibody response to a variety of antigens requires thymus dependent cells for its induction (Lance, 1970; Miller & Osoba, 1967). Neve~heless, there have been no reports on the correlation of thymus with antibody production in H. nuna infection, nor the effect of transferring sensitized cells. Therefore it can not be concluded that cell-mediated immunity is solely the mediator of protection (Okamoto & Koizumi, 1972), although it seems probable that it may be involved. It is therefore important to identify the factors involved in the passive transfer of immunity. As the index of protection in H. nana infection, the reduction in the number of cysticercoids 4 days post challenge has been used (Hunninen, 1935). However, the index is an oversimpli~cation of the events which occur in the intestine, since it provides no information as to whether the lethal effect was directed against the egg, the oncosphere in the intestinal lumen, the oncosphere in the villus, or the early post-oncospheral phase in the villus (Gemmell & Macnamara, 1972). It is therefore very important to identify the stage at which protection occurs in actively or passively immunized hosts (Bailey, 1951;
INTRODUCTION Hymenolepis l~ana elicits protective immunity against reinfection following a primary egg infection in mice (Hearin, 1941; Heyneman, 1962), but the precise m~hanism of immLlnity against H. nuna is unknown (Gemmell & Macnamara, 1972; Larsh, 1951; Larsh & Weatherly, 1975; Weinmann, 1966, 1970). The humoral basis of protective immunity against H. nanu reinfection was studied by Hearin (1941) and Larsh (1942) who showed serum and milk from infected donor mice could transfer the immunity to uninfected mice, whereas Weinmann (1966) reported inconsistent results. IX Conza (1969) reported protective action of passively transferred immune serum and immunoglubolin fractions against shellfree eggs injected subcutaneously. Although he found that serum inhibited larval differentiation in ectopic tissue, he demonstrated no evidence that serum antibodies serve to resist an egg challenge in the intestine: ii is unknown whether the onwsphere in ectopic tissue was the same as that in the lumen before invasion or that in the villus after invasion, Therefore the protective role of immune serum can not be said to have conclusively been demonstrated. On the other hand, cell-mediated immunity has been suggested to be involved because protective 67
68
1.J.P. V”,
A. I ro
IX ConLa, 1969; Ito & Yaniamoto fin press); Weinmann, 1966). Recently it was reported that antibodies against hatched oncospheres, which have been considered immunogenic (Di Conza, 1969; Gemmell & Macnamara, 1972; Weinmann, 1966), were demonstrable it? V&U in immune mouse, and rabbit serum (Ito, 1975). In the present study, the action of immune serum in viva has been studied by serum transfer experiments. MATERIALS
AND METHODS
Parasites. The preparation of shell-free eggs of H. nuna (Berntzen & Voge, 1965) was previously described (Okamoto, 1968; Ito, 1975). Throughout the text the word egg means a shell-free egg of H. ~runu. Animals. Worm-free, laboratory-raised albino mice of dd strain (random-bred) were used. Uninfected mice of both sexes born from uninfected mothers and weaned when 18-21 days old were used both as donors and as recipients. The donors of nonimmune serum and the recipients were kept in wjre-bottomed plastic cages, 8-10 mice per cage. The donors of immune serum were kept on bedding in plastic cages, 5-7 litters of the same age per cage. All the cages were kept clean by renewing the wire-bottom or the bedding every day. In all the experiments, 4- to 5-week-old mice were used as the recipients. Preparation of serum. A total of 2000 donors were immunized by infection with 500 eggs of H. nun0 when 3 weeks old. After 2-10 weeks immune serum was taken
by cardiac puncture. Tw[~-week-serulll in the text mean\ pooled serum from mice immunized for 2 weeks. Nonimmune sera were prepared from Gweek-old uninfected 20°C without mice. All pooled sera were stored at preservatives and inactivated at 5fi”C for 30 min before use. Mar&r antibody. Serum agglutinins against hatched ontospheres of N. 11ana were titrated by the method of lto (1975). In experiment 1, ten pools of S-week-serum were used as immune serum. The titre of three of the ten pools was 32 and the seven others 64; that of nonimmune serum was 0. Sera other than those used in experiment I were not tested. Immunization andassay ofpu~tection. In all experiments serum was injected intraperitoneally. Details of the sera used and the days on which serum was injected are given in the descriptions of each experiment. Two hours after injection on day 0, each mouse was infected with about 2000 eggs of H. nana. In the experiments other than experiment 3B, all the mice were killed on day 4 (94.-98 h after infection) and fully developed cysticercoid burdens determined. The criterion for assessing transfer of protection was whether or not cysticercoids with adult hooks on the rostellum were present in the ~lltestinal villi (Hunninen, 1935). In experiment 3A, earlier larval stages in the villi were observed and counted. In experiment 3B, the presence of adult tapeworms on day II was also determined. Stcztistical analysis. Student’s t test was used to determine the statistical singificance of the observed differences in numbers of parasites recovered from the experimental and control groups. A probability greater than 0.05 was not considered significant.
TABLE l-EFFECTOFVARYINGTHEVOLIJMEAND
Schedule of injections
No. of nools (Titre*) tested
7. IV77
FREQ~~~~YO~SERUMINJECTIONS
No. of micelgp infected with cysticercoids day 4
No. of cysticercoids recovered dav 4 Mean .i: s.~.(t&ge)
._
6 Injections (days-4 to I) 0.5 ml or 0.2 ml/day Immune serum Nonimmune serum 0.85 % NaCl No treatment
Total volume serum 3.0 ml
1 (32) 5 (64) 2 ( 0) __
I.2 ml 6.5; 6.4 (2 -16) NT 5/Y 3863 1-90.0 NT NT
l/S 1 Sj28 96& 175 (l-45) If/II 399.9.+1355 l3/13 426.3$141*4 I l/l 1 3950mt200.6
4:5
Total volume serum
3 Injections (days-2 to 0) I.0 ml or 0.5 ml/day
I .5 ml
3.0 ml
Immune serum
1 (3.2) 1 (64)
Nonimmune serum No treatment
1 ( 0)
I Injection (day-l) 3.0 ml, 2.0 ml or 1.O ml/day Immune serum 1 (32) I (64) Nonimmune serum 1 ( 0) No treatment
l/5 3 215 6.5;!- 2.1 5/S 397.8,L 124.5 515 353.4$ 82.6
3.0 ml
w I!5 h/6 6/6
80 -. 5.7 2 259.1 i 276*8f
*Titre: titre of agglutinins against the oncosphere
82.2 71.2
51.5 36.2; 15.2 (19-53) NT NT 5i’S 249.0 i-22.6 Total volume serum 2.0 ml 415 54.8% 736@162) NT NT 5/s 24l.O:L_1003
of H. rtana.
I.0 ml 97.2189.8 (46-278) NT NT 616 307.3 3165.9 616
I.J.P. VOL.
Passive protection against H. nanu
7. 1971
almost completely immune. Neither O-week-serum nor 2-week-serum transferred protection.
RESULTS Experiment
I. The eflect of serum tram&r
A total of 152 mice were used, half of which were injected with immune serum. The details and the results are shown in Table 1. Mice given immune serum @week-serum) showed a tendency to resist an egg infection; most of the mice receiving a total of 3.0 ml of immune serum of all the ten pools were not infected with cysticercoids in the villi, whereas almost all the mice injected with 52.0 ml of immune serum did not completely resist the egg challenge, but some mice harboured significantly fewer cysticercoids than the controls. There appeared to be no correlation between protective potency and oncospheral agglutinin titre in the pools of serum used. All 78 of the controls were heavily infected; 27 mice receiving nonimmune serum were infected with the same level of cysticercoid burdens as the 13 mice injected with 0*85’% NaCl and the 38 untreated controls. Accordingly, the control mice in the following experiments were untreated. Experiment 2. The effect of varying the period of immunization of serum donors
Seven pools of serum were used : 0-, 2-, 3-, 4-, S-,7-, and lo-week-serum and the time course of development of protective antibodies was examined by counting the average number of cysticercoids from 8 to 10 mice given 1.Oml of serum/day for 3 days (days -2 to 0). The results (Table 2) show that protection was transferred to the recipients given serum taken after 3 or more weeks of immunization. When receiving 4- to 7-week-serum, the recipients became
TABLE Z-TIME
COURSE OF PROTECTIVE ANTIBODY
_~ Group infected O-Week-serum Control 2-Week-serum Control 3-Week-serum Control 4-Week-serum Control 5-Week-serum Control ‘I-Week-serum Control 1O-Week-serum Control
Experiment
No. of mice found Mean
3. Time of action of immune serum
Mice receiving 5-week-serum (1.0 ml/day from day-2 to day 0) were examined on day 4 to see whether they harboured any early stage larvae in the villi. Nine of the 10 experimental mice injected with serum obtained the earlier stage II or III larvae of Voge & Heyneman (1957) in the villi. The remaining one mouse harboured a small number of cysticercoids in addition to stage IL or III larvae. No stage II or III larvae were observed in any untreated mice on day 4 and the number of the stage II or III larvae, 254.1 f22.7 (S.D.),was similar to that of cysticercoids in the other ten untreated controls, 273.5 k70.3. 3B. An additional experiment was performed to see if stage 11 or III larvae in the villi were able to develop to cysticercoids and grow to adult tapeworms. Although cysticercoids in each of the 20 control mice grew to adult tapeworms (2300 worms) in 11 days post infection, the stage II or 111 larvae in 14 of the 20 experimental mice given 1.0 ml/day of S-week-serum from day -2 to day 0, did not grow to adult tapeworms; neither cysticercoids in the villi nor adult tapeworms in the lumen existed in the 14 mice. In the other six of the 20 mice, although significantly fewer than control, were some adult tapeworms found (I, 10, 18, 30,43 or 62 worms). 3A.
Experiment 4. Site of action of immune serum
The degree of protection was examined by giving 3.0 ml of 7-week-serum by one injection before or
PRODUCTION
SHELL-FREE EGGSOF
lo/lo 919 lo;10 lo/lo $9 919 118 lOjl0 2/8 818 4/10 IO/l0 IO/l0 lOi
69
IN MICE FOLLOWING
A PRIMARY
INFECTIONWITH
Hymenolepisnana No. of cysticercoids Statistical in infected mice significance* Mean S.D. -... .-.. __~~~_~_~ 174.2 NS 3700 226.5 357.0 976 NS 352.0 101.1 397.3 11.6 -=o.ooi 12.4 206.4 4176 P.
43i.o 3 305,6 28z.6
113.1 2 78.2 97.5 3
67.3 379.7
41.1 65.9
-=OOOl
* Mean of experimental group vs. control mean. NS: not significant. The effect of serum transfer was examined on day 4 by counting the number of cysticercoids in mice given serum (I .Omliday from day-2 to day O), infected with 2000 shell-free eggs on day 0.
500
.A. I10
70
after an egg infection and counting the number of cysticercoids. The details and the resutt are shown in Table 3. The serum had a significantly protective TABLE 3-NUMBEROF CYSTICERCOIDS RECOVEREDON DAY WHEN IMMUNE SERUM (3’0 ml) WAS ADMINISTERED BY ONE INJECTION BEFORE OR AFTER AN INFECTION WITH 2000 SHELL-FREE EGGS OF Hynrenoleph trannON DAY 0
4
___ _-
Group
Day when serum was given
No. of mice found infected
No. of cysticercoids in infected mice Mean S.D.
2 2/s 3 A day I 30.2 19.9 B day 0 5;s 515 696 58.3 c day 1 D 5:5 455.2 64.8 day 2 225.6 E 513.0 N.T. 515 __-_~~ ~_. B or C vs. D and E: P < 0.01, B vs. C: not significant (NS), D vs. E: NS. N.T., no treatment. All groups consisted of 5 mice. effect in three (A, B, and Cl of the four groups of recipients, but only when immune serum was given on day -1 (group A), did the mice become almost completely immune; three of the five mice were not infected with cysticercoids in the villi. When serum was given either on day 0 (group B) or on day 1 (group C), mice became immune (PC 0.01) but in neither group was the level of immunity transferred comparable to that of group A. When receiving the serum on day 2 (group D), the recipients showed no immunity. DISCUSSION The results confirm Hearin’s (1941) results and show clearly that it is possible to transfer immunity against the cestode Hymenolepis nann with serum taken from actively immunized mice. In mice given immune serum, oncospheres hatched, invaded the intestinal villi and differentiated to stage II or III larvae (Voge & Heyneman, 1957), but almost all of them failed to differentiate to cysticercoids by 4 days post infection. The stage II or III larvae, perhaps earlier dead within 4 days post infection, did not grow to cysticercoids nor adult tapeworms in 1 I days post infection in the majority of the mice. Most of the oncospheres might have been damaged during the invasion stage as suggested by Di Conza (1969) and Weinmann (1966). Nevertheless, the oncospheres did invade the villi. Passive protection was induced by giving serum to recipients not only prior to the oncospheral invasion but also post the active invasion. It is suggested that the susceptibility of the oncospheres to ~nlnlune serum changed in the intestinal villi between days I and 2, and the lethal effect of serum was directed against the step of reorganization of the oncospheres in the tissue. As the immune serum contained agglutinating
I.J.P. VOI
7.
1977
antibodies against the oncospheres and since the time course of development of protective antibodies in the serum was similar to that of the agglutinating antibodies (lto, 1975). it seems probable that the development of the oncosphere is damaged by direct action of these antibodies. The major action of immune serum is therefore likely to be damaging hatched oncospheres in both the intestinal lumen and the villi. In 52 of a total of 77 mice immunized actively with various doses of eggs (552000 eggs), only a small number of oncospheres were found in the villi 4 days post challenge with 2000 eggs. In 23 others no cysticercoids or oncospheres were found, and the remaining two harboured a small number of cysticercoids (Ito & Yamamoto (in press)). This agrees basically with Bailey’s (1950, Heyneman’s (l962), or Weinmann’s (1966) observation that early stage larvae were found in the villi of some actively immunized hosts, but no cysticercoids formed in these animals. Weinmann (1966) found a striking influence of mucosal extract of actively immunized hosts on immature worms (but not the oncospheres) in vitro. Therefore the degree of protection conferred by serum transfer seems to be similar to, but slightly weaker than that stimulated by active immunization. Weinmann (1966) explained that his inconsistent results of passive protection were due to the loss of the antibodies which might be absorbed or fixed to host tissue. The fact that the volume of serum transferred is so critical, however, seems to be difficult to explain at this time but may suggest a requirement for sensitized cells in active immunization. On the other hand, it seems probable that immune serum contains antibodies against older parasite; cysticercoid and/or adult tapeworm (Coleman, Carty & Graziardei, 1968). Nevertheless, the highly immunogenic parasite which has made the host immune against reinfection within 1-2 days post an egg infection (Hearin, 1941; Heyneman, 1962) completes its development in the imnlunized host. Passive protection against tailless cysticercoids, which were prepared from the mouse intestine viili (lto, in press), has been unsuccessful by serum transfer [unpublished). This agrees with the present result of Experiment 4, and may also imply that after a certain point cysticercoids are unaffected by antibodies (Musoke &Williams, 1975). It is therefore very interesting to know the means whereby older parasite other than the oncosphere or the early post-oncospheral phase of H. nana evades the host response. ,4ck~u~le~~~ments-I wish to thank Mrs. Mieko Yamamoto and Miss Masako Sato for their technical assistance. I am also indebted to Professor M. Yokogawa, Chiba University._I Jaoan for his critical reviewing and rewriting . of the English manuscript. This work was submitted in partial fulfilment of the requirements for the Ph.D. thesis of the Tohoku University, Sendai, Japan.
I.J.P. VOL.
71
Passive protection against H. nana
7. 1977
mice inoculated with different doses of shell-free eggs.
REFERENCES
Japanese Journal of Parasitology.
BAILEYW. S. 1951. Host-tissue reactions to initial and superimposed infections with Hymenolepis nana var. fraterna.
Journal
of Parasitology
37: 44G-444.
BERNTZENA. K. & V~GE M. 1965. In vitro hatching of oncospheres of four hymenolepidid cestodes. Journal of Parasitology
51: 235-242.
COLEMANR. M., CARTYJ. M. & GRAZIARDEIW. D. 1968. Immunogenicity and phylogenic relationship of tapeworm antigens produced by Hymenolepis nana and Hymenolepis
dimunita.
Immunology
15: 297-304.
Dr CONZA J. J. 1969. Protective action of passively transferred immune serum and immunoglobulin fractions against tissue invasive stages of the dwarf tapeworm, Hymenolepis nana. Experimental Parasitology 25: 368-375.
GEMMELLM. A. & MACNAMARAF. N. 1972. Immune response to tissue parasites II. Cestodes. In Immunity to Animal Parasites (Edited by ~SOULSBY E. J. L.) pp. 236272. Academic Press, New York. HEARINJ. T. 1941. Studies on the acquired immunity to the dwarf tapeworm Hymenolepis nana var. fraterna, in the mouse host. American Journal of Hygiene 33: 71-87.
HEYNEMAND. 1962. Studies on helminth immunity IV. Rapid onset of resistance by the white mouse against a challenging infection with eggs of Hymenolepis nana (Cestoda Hymenolepididae). Journal of Immunology 88: 210-220.
HUNNINEN A. V. 1935. A method of demonstrating cysticercoids of Hymenolepis fraterna (H. nana var. fraterna Stiles) in the intestinal villi of mice. Journal of Parasitology
21: 124-125.
A. 1975. In vitro oncospheral agglutination given by immune sera from mice infected, and rabbits injected, with eggs of Hymenolepis nana. Parasitology 71:
IT0
465473.
A. In press. A simple method for collecting infective cysticercoids of Hymenolepis nanu from the mouse intestine. Journal of Parasitology. ITO A. & YAMAMOTO M. In press. The mode of active protection against Hymenloepis nana reinfection in
ITO
LANCE M. 1970. The selective action of antilymphocyte serum on recirculating lymphocytes. A review of the evidence and alternatives. Clinical and Experimental Immunology
6: 789-802.
LARSHJ. E. 1942. Transmission from mother to offspring of immunity against the mouse cestode, Hymenolepis nana var. fraterna. American Journal of Hygiene 36: 187-194.
LARSHJ. E. 1951. Host-parasite relationships in cestode infections, with emphasis on host resistance. Journal of Parasitology
37: 343-352.
LARSH J. E. & WEATHERLYN. E. 1975. Cell-mediated immunity against certain parasitic worms. In Advances in Parasitology (Edited by DAWES B.). Vol. 13, pp. 183-222. Academic Press, London. MILLERJ. F. A. P. & OSOBA D. 1967. Current conceots of the immunological function of the thymus. Pkysiological Reviews
47: 437-520.
MUSOKEA. J. & WILLIAMSJ. F. 1975. The immunological response of the rat to infection with Taeniu taeniaefbrmis V. Sequence of appearance of protective immunoglobulins and the mechanism of action of 7Sy2a antibodies. Immunology 29: 855-866. OKAMOTOK. 1968. Effect of noenatal thymectomy on acquired resistance to Hymenolepis nana in mice. Japanese Journal of Parasitology
17: 53-59.
OKAMOTOK. & KOIZUMI M. 1972. Hymenolepis nana: Effect of antithymocyte serum on acquired immunity in mice. Experimental Parasitology 32: 56-61. VOGE M. & HEYNEMAND. 1957. Development of Hymenolepis nana and Hymenolepis diminuta (Cestoda: Hymenolepididae) in the intermediate host Tribolirrm confusum. University zoology 59: 545-579.
of
California
Publication
in
WEINMANNC. J. 1966. Immunity mechanism in cestode infection. In Biology of Parasites (Edited by SOULSBY E. J. L.) pp. 301-320. Academic Press, New York. WEINMANNC. J. 1970. Immunity in mammalian hosts. In Immunity to Parasitic Animals (Edited by JACKSON G. J., HERMANR. &SINGER I.), Vol. II, pp. 1021-1059. Appleton, New York.
Journal
for
Parasitology.
1977.
Vol. 7. pp. 67-71.
Pergamon
Press. Printed
in Great
Britain.
THE MODE OF PASSIVE PROTECTION AGAINST ~Y~~~Q~~PI~ NANA INDUCED BY SERUM TRANSFER A. IT0 Department
of Medical Biology, School of Medicine, Showa University, Hatanodai, Shinagawaku, Tokyo, Japan (Received 23 February 1976; in revised form 6 July 1976)
A. 1977. The mode of passive protection against Hymenu~ep~s nana induced by serum transfer. international ~uur~~ for Paru~~to~~gy 7: 67-71. A protective immunity against the cestode ~ymeno~epis nana was transferred with serum taken from actively immunized mice. All of 17 pooled sera examined, which were taken from mice immunized for 3 or more weeks, were strongly effective. Intraperitoneal injection of a total of 3.0 ml serum made the recipient mice (44 weeks old) almost completely immune. In almost all the mice given immune serum no cysticercoids were found on day 4. In mice receiving immune serum, oncospheres hatched, invaded the intestinal villi and differentiated to stage II or III larvae, but failed to develop to fully developed cysticercoids. The degree of protection conferred by serum transfer was similar to, but slightly weaker than that stimulated by active immuni~tion. The major effect of immune serum was damaging hatched oncospheres in both the intestinal lumen and the villi within 1 day post infection. Abstract-ITo
INDEX KEY WORDS: oncosphere; cysticercoid.
Hymenolepis
nana;
mice; intestine;
immunity;
serum transfer;
immunity against H. nann reinfection is thymus dependent (Okamoto, 1968; Okamoto & Koizumi, 1972). However, it is well-known that the antibody response to a variety of antigens requires thymus dependent cells for its induction (Lance, 1970; Miller & Osoba, 1967). Neve~heless, there have been no reports on the correlation of thymus with antibody production in H. nuna infection, nor the effect of transferring sensitized cells. Therefore it can not be concluded that cell-mediated immunity is solely the mediator of protection (Okamoto & Koizumi, 1972), although it seems probable that it may be involved. It is therefore important to identify the factors involved in the passive transfer of immunity. As the index of protection in H. nana infection, the reduction in the number of cysticercoids 4 days post challenge has been used (Hunninen, 1935). However, the index is an oversimpli~cation of the events which occur in the intestine, since it provides no information as to whether the lethal effect was directed against the egg, the oncosphere in the intestinal lumen, the oncosphere in the villus, or the early post-oncospheral phase in the villus (Gemmell & Macnamara, 1972). It is therefore very important to identify the stage at which protection occurs in actively or passively immunized hosts (Bailey, 1951;
INTRODUCTION Hymenolepis l~ana elicits protective immunity against reinfection following a primary egg infection in mice (Hearin, 1941; Heyneman, 1962), but the precise m~hanism of immLlnity against H. nuna is unknown (Gemmell & Macnamara, 1972; Larsh, 1951; Larsh & Weatherly, 1975; Weinmann, 1966, 1970). The humoral basis of protective immunity against H. nanu reinfection was studied by Hearin (1941) and Larsh (1942) who showed serum and milk from infected donor mice could transfer the immunity to uninfected mice, whereas Weinmann (1966) reported inconsistent results. IX Conza (1969) reported protective action of passively transferred immune serum and immunoglubolin fractions against shellfree eggs injected subcutaneously. Although he found that serum inhibited larval differentiation in ectopic tissue, he demonstrated no evidence that serum antibodies serve to resist an egg challenge in the intestine: ii is unknown whether the onwsphere in ectopic tissue was the same as that in the lumen before invasion or that in the villus after invasion, Therefore the protective role of immune serum can not be said to have conclusively been demonstrated. On the other hand, cell-mediated immunity has been suggested to be involved because protective 67
68
1.J.P. V”,
A. I ro
IX ConLa, 1969; Ito & Yaniamoto fin press); Weinmann, 1966). Recently it was reported that antibodies against hatched oncospheres, which have been considered immunogenic (Di Conza, 1969; Gemmell & Macnamara, 1972; Weinmann, 1966), were demonstrable it? V&U in immune mouse, and rabbit serum (Ito, 1975). In the present study, the action of immune serum in viva has been studied by serum transfer experiments. MATERIALS
AND METHODS
Parasites. The preparation of shell-free eggs of H. nuna (Berntzen & Voge, 1965) was previously described (Okamoto, 1968; Ito, 1975). Throughout the text the word egg means a shell-free egg of H. ~runu. Animals. Worm-free, laboratory-raised albino mice of dd strain (random-bred) were used. Uninfected mice of both sexes born from uninfected mothers and weaned when 18-21 days old were used both as donors and as recipients. The donors of nonimmune serum and the recipients were kept in wjre-bottomed plastic cages, 8-10 mice per cage. The donors of immune serum were kept on bedding in plastic cages, 5-7 litters of the same age per cage. All the cages were kept clean by renewing the wire-bottom or the bedding every day. In all the experiments, 4- to 5-week-old mice were used as the recipients. Preparation of serum. A total of 2000 donors were immunized by infection with 500 eggs of H. nun0 when 3 weeks old. After 2-10 weeks immune serum was taken
by cardiac puncture. Tw[~-week-serulll in the text mean\ pooled serum from mice immunized for 2 weeks. Nonimmune sera were prepared from Gweek-old uninfected 20°C without mice. All pooled sera were stored at preservatives and inactivated at 5fi”C for 30 min before use. Mar&r antibody. Serum agglutinins against hatched ontospheres of N. 11ana were titrated by the method of lto (1975). In experiment 1, ten pools of S-week-serum were used as immune serum. The titre of three of the ten pools was 32 and the seven others 64; that of nonimmune serum was 0. Sera other than those used in experiment I were not tested. Immunization andassay ofpu~tection. In all experiments serum was injected intraperitoneally. Details of the sera used and the days on which serum was injected are given in the descriptions of each experiment. Two hours after injection on day 0, each mouse was infected with about 2000 eggs of H. nana. In the experiments other than experiment 3B, all the mice were killed on day 4 (94.-98 h after infection) and fully developed cysticercoid burdens determined. The criterion for assessing transfer of protection was whether or not cysticercoids with adult hooks on the rostellum were present in the ~lltestinal villi (Hunninen, 1935). In experiment 3A, earlier larval stages in the villi were observed and counted. In experiment 3B, the presence of adult tapeworms on day II was also determined. Stcztistical analysis. Student’s t test was used to determine the statistical singificance of the observed differences in numbers of parasites recovered from the experimental and control groups. A probability greater than 0.05 was not considered significant.
TABLE l-EFFECTOFVARYINGTHEVOLIJMEAND
Schedule of injections
No. of nools (Titre*) tested
7. IV77
FREQ~~~~YO~SERUMINJECTIONS
No. of micelgp infected with cysticercoids day 4
No. of cysticercoids recovered dav 4 Mean .i: s.~.(t&ge)
._
6 Injections (days-4 to I) 0.5 ml or 0.2 ml/day Immune serum Nonimmune serum 0.85 % NaCl No treatment
Total volume serum 3.0 ml
1 (32) 5 (64) 2 ( 0) __
I.2 ml 6.5; 6.4 (2 -16) NT 5/Y 3863 1-90.0 NT NT
l/S 1 Sj28 96& 175 (l-45) If/II 399.9.+1355 l3/13 426.3$141*4 I l/l 1 3950mt200.6
4:5
Total volume serum
3 Injections (days-2 to 0) I.0 ml or 0.5 ml/day
I .5 ml
3.0 ml
Immune serum
1 (3.2) 1 (64)
Nonimmune serum No treatment
1 ( 0)
I Injection (day-l) 3.0 ml, 2.0 ml or 1.O ml/day Immune serum 1 (32) I (64) Nonimmune serum 1 ( 0) No treatment
l/5 3 215 6.5;!- 2.1 5/S 397.8,L 124.5 515 353.4$ 82.6
3.0 ml
w I!5 h/6 6/6
80 -. 5.7 2 259.1 i 276*8f
*Titre: titre of agglutinins against the oncosphere
82.2 71.2
51.5 36.2; 15.2 (19-53) NT NT 5i’S 249.0 i-22.6 Total volume serum 2.0 ml 415 54.8% 736@162) NT NT 5/s 24l.O:L_1003
of H. rtana.
I.0 ml 97.2189.8 (46-278) NT NT 616 307.3 3165.9 616
I.J.P. VOL.
Passive protection against H. nanu
7. 1971
almost completely immune. Neither O-week-serum nor 2-week-serum transferred protection.
RESULTS Experiment
I. The eflect of serum tram&r
A total of 152 mice were used, half of which were injected with immune serum. The details and the results are shown in Table 1. Mice given immune serum @week-serum) showed a tendency to resist an egg infection; most of the mice receiving a total of 3.0 ml of immune serum of all the ten pools were not infected with cysticercoids in the villi, whereas almost all the mice injected with 52.0 ml of immune serum did not completely resist the egg challenge, but some mice harboured significantly fewer cysticercoids than the controls. There appeared to be no correlation between protective potency and oncospheral agglutinin titre in the pools of serum used. All 78 of the controls were heavily infected; 27 mice receiving nonimmune serum were infected with the same level of cysticercoid burdens as the 13 mice injected with 0*85’% NaCl and the 38 untreated controls. Accordingly, the control mice in the following experiments were untreated. Experiment 2. The effect of varying the period of immunization of serum donors
Seven pools of serum were used : 0-, 2-, 3-, 4-, S-,7-, and lo-week-serum and the time course of development of protective antibodies was examined by counting the average number of cysticercoids from 8 to 10 mice given 1.Oml of serum/day for 3 days (days -2 to 0). The results (Table 2) show that protection was transferred to the recipients given serum taken after 3 or more weeks of immunization. When receiving 4- to 7-week-serum, the recipients became
TABLE Z-TIME
COURSE OF PROTECTIVE ANTIBODY
_~ Group infected O-Week-serum Control 2-Week-serum Control 3-Week-serum Control 4-Week-serum Control 5-Week-serum Control ‘I-Week-serum Control 1O-Week-serum Control
Experiment
No. of mice found Mean
3. Time of action of immune serum
Mice receiving 5-week-serum (1.0 ml/day from day-2 to day 0) were examined on day 4 to see whether they harboured any early stage larvae in the villi. Nine of the 10 experimental mice injected with serum obtained the earlier stage II or III larvae of Voge & Heyneman (1957) in the villi. The remaining one mouse harboured a small number of cysticercoids in addition to stage IL or III larvae. No stage II or III larvae were observed in any untreated mice on day 4 and the number of the stage II or III larvae, 254.1 f22.7 (S.D.),was similar to that of cysticercoids in the other ten untreated controls, 273.5 k70.3. 3B. An additional experiment was performed to see if stage 11 or III larvae in the villi were able to develop to cysticercoids and grow to adult tapeworms. Although cysticercoids in each of the 20 control mice grew to adult tapeworms (2300 worms) in 11 days post infection, the stage II or 111 larvae in 14 of the 20 experimental mice given 1.0 ml/day of S-week-serum from day -2 to day 0, did not grow to adult tapeworms; neither cysticercoids in the villi nor adult tapeworms in the lumen existed in the 14 mice. In the other six of the 20 mice, although significantly fewer than control, were some adult tapeworms found (I, 10, 18, 30,43 or 62 worms). 3A.
Experiment 4. Site of action of immune serum
The degree of protection was examined by giving 3.0 ml of 7-week-serum by one injection before or
PRODUCTION
SHELL-FREE EGGSOF
lo/lo 919 lo;10 lo/lo $9 919 118 lOjl0 2/8 818 4/10 IO/l0 IO/l0 lOi
69
IN MICE FOLLOWING
A PRIMARY
INFECTIONWITH
Hymenolepisnana No. of cysticercoids Statistical in infected mice significance* Mean S.D. -... .-.. __~~~_~_~ 174.2 NS 3700 226.5 357.0 976 NS 352.0 101.1 397.3 11.6 -=o.ooi 12.4 206.4 4176 P.
43i.o 3 305,6 28z.6
113.1 2 78.2 97.5 3
67.3 379.7
41.1 65.9
-=OOOl
* Mean of experimental group vs. control mean. NS: not significant. The effect of serum transfer was examined on day 4 by counting the number of cysticercoids in mice given serum (I .Omliday from day-2 to day O), infected with 2000 shell-free eggs on day 0.
500
.A. I10
70
after an egg infection and counting the number of cysticercoids. The details and the resutt are shown in Table 3. The serum had a significantly protective TABLE 3-NUMBEROF CYSTICERCOIDS RECOVEREDON DAY WHEN IMMUNE SERUM (3’0 ml) WAS ADMINISTERED BY ONE INJECTION BEFORE OR AFTER AN INFECTION WITH 2000 SHELL-FREE EGGS OF Hynrenoleph trannON DAY 0
4
___ _-
Group
Day when serum was given
No. of mice found infected
No. of cysticercoids in infected mice Mean S.D.
2 2/s 3 A day I 30.2 19.9 B day 0 5;s 515 696 58.3 c day 1 D 5:5 455.2 64.8 day 2 225.6 E 513.0 N.T. 515 __-_~~ ~_. B or C vs. D and E: P < 0.01, B vs. C: not significant (NS), D vs. E: NS. N.T., no treatment. All groups consisted of 5 mice. effect in three (A, B, and Cl of the four groups of recipients, but only when immune serum was given on day -1 (group A), did the mice become almost completely immune; three of the five mice were not infected with cysticercoids in the villi. When serum was given either on day 0 (group B) or on day 1 (group C), mice became immune (PC 0.01) but in neither group was the level of immunity transferred comparable to that of group A. When receiving the serum on day 2 (group D), the recipients showed no immunity. DISCUSSION The results confirm Hearin’s (1941) results and show clearly that it is possible to transfer immunity against the cestode Hymenolepis nann with serum taken from actively immunized mice. In mice given immune serum, oncospheres hatched, invaded the intestinal villi and differentiated to stage II or III larvae (Voge & Heyneman, 1957), but almost all of them failed to differentiate to cysticercoids by 4 days post infection. The stage II or III larvae, perhaps earlier dead within 4 days post infection, did not grow to cysticercoids nor adult tapeworms in 1 I days post infection in the majority of the mice. Most of the oncospheres might have been damaged during the invasion stage as suggested by Di Conza (1969) and Weinmann (1966). Nevertheless, the oncospheres did invade the villi. Passive protection was induced by giving serum to recipients not only prior to the oncospheral invasion but also post the active invasion. It is suggested that the susceptibility of the oncospheres to ~nlnlune serum changed in the intestinal villi between days I and 2, and the lethal effect of serum was directed against the step of reorganization of the oncospheres in the tissue. As the immune serum contained agglutinating
I.J.P. VOI
7.
1977
antibodies against the oncospheres and since the time course of development of protective antibodies in the serum was similar to that of the agglutinating antibodies (lto, 1975). it seems probable that the development of the oncosphere is damaged by direct action of these antibodies. The major action of immune serum is therefore likely to be damaging hatched oncospheres in both the intestinal lumen and the villi. In 52 of a total of 77 mice immunized actively with various doses of eggs (552000 eggs), only a small number of oncospheres were found in the villi 4 days post challenge with 2000 eggs. In 23 others no cysticercoids or oncospheres were found, and the remaining two harboured a small number of cysticercoids (Ito & Yamamoto (in press)). This agrees basically with Bailey’s (1950, Heyneman’s (l962), or Weinmann’s (1966) observation that early stage larvae were found in the villi of some actively immunized hosts, but no cysticercoids formed in these animals. Weinmann (1966) found a striking influence of mucosal extract of actively immunized hosts on immature worms (but not the oncospheres) in vitro. Therefore the degree of protection conferred by serum transfer seems to be similar to, but slightly weaker than that stimulated by active immunization. Weinmann (1966) explained that his inconsistent results of passive protection were due to the loss of the antibodies which might be absorbed or fixed to host tissue. The fact that the volume of serum transferred is so critical, however, seems to be difficult to explain at this time but may suggest a requirement for sensitized cells in active immunization. On the other hand, it seems probable that immune serum contains antibodies against older parasite; cysticercoid and/or adult tapeworm (Coleman, Carty & Graziardei, 1968). Nevertheless, the highly immunogenic parasite which has made the host immune against reinfection within 1-2 days post an egg infection (Hearin, 1941; Heyneman, 1962) completes its development in the imnlunized host. Passive protection against tailless cysticercoids, which were prepared from the mouse intestine viili (lto, in press), has been unsuccessful by serum transfer [unpublished). This agrees with the present result of Experiment 4, and may also imply that after a certain point cysticercoids are unaffected by antibodies (Musoke &Williams, 1975). It is therefore very interesting to know the means whereby older parasite other than the oncosphere or the early post-oncospheral phase of H. nana evades the host response. ,4ck~u~le~~~ments-I wish to thank Mrs. Mieko Yamamoto and Miss Masako Sato for their technical assistance. I am also indebted to Professor M. Yokogawa, Chiba University._I Jaoan for his critical reviewing and rewriting . of the English manuscript. This work was submitted in partial fulfilment of the requirements for the Ph.D. thesis of the Tohoku University, Sendai, Japan.
I.J.P. VOL.
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Passive protection against H. nana
7. 1977
mice inoculated with different doses of shell-free eggs.
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in
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