Schistosoma mansoni: The attrition of a challenge infection in mice immunized with highly irradiated live cercariae

EXPERIMENTAL

PARASITOLOoY

Schistosoma

50,

212-221

(1980)

mansoni: The Attrition of a Challenge Infection Immunized with Highly Irradiated Live Cercariae KATHLEEN Division

of

Parasitology, London

(Accepted

L. MILLER National NW7 IAA,

AND

Institute England,

for publication

S.

R.

SMITHERS

for

Medical Resewrch, United Kingdom

23 November

in Mice

Mill

Hill,

1979)

MILLER, K. I., AND SMITHERS, S. R. 1980. Schistosoma mansonic The attrition of a challenge infection in mice immunized with highly irradiated live cercariae. Experimental Parasitology 50, 212-221. Mice immunized percutaneously with 400 Schistosoma mansoni cercariae given 20 kR of Vo irradiation were shown to develop an immunity in which nearly 80% of the parasites that would be expected to survive in control mice were killed. The major attrition of parasites was shown to occur within the first 4 days after challenge. Marked differences in the number of parasites which were recovered from the skin of immune mice and the failure of the majority of parasites to reach the lungs of immune mice indicated that the major site of attrition was in the skin. A further trickle of parasite deaths was evident beyond Day 5, but after Day 14 no further attrition of parasites appeared to occur. Mice immunized in the abdominal skin demonstrated similar levels of immunity whether challenged in the abdominal skin or in the ear. Immunization intramuscularly with irradiated schistosomula induced a much lower level of resistance and the marked parasite attrition in the skin at Day 2 was absent. Immunization with only 50 irradiated cercariae was shown to induce a level of skin immunity equivalent to that seen with 400 irradiated cercariae. The majority of cercariae given 20 kR of 6oCo irradiation remained in the skin; approximately 2% only reached the lungs. These studies demonstrate that percutaneous immunization of mice with highly irradiated cercariae induced a strong immunity which was largely effective in the skin. This immunity differed from that developed by chronically infected mice where the major attrition of parasites occurs after the lung phase of migration. The results also suggest that the penetration or persistence in the skin of live attenuated schistosomula may play a crucial role in the induction of a high level of skin immunity. INDEX DESCRIPTORS: Schistosorna rnansoni; Trematode; Irradiated cercariae; Mouse; Skin; Immunity.

INTRODUCTION

Mice infected with Schistosoma mansoni develop concomitant immunity where the worms of a primary infection persist to give a chronic infection, while the majority of the parasites of a reinfection are killed during their early migration (Doenhoff, ef al. 1978). Experiments in which the worms of a challenge infection have been recovered from chronically infected mice have demonstrated two distinct phases of parasite attrition. An “early phase” occurs within the first 3 days of exposure and accounts for the death of approximately 30% of the infection which would survive in control mice. This phase probably occurs in the 212 0014-4894/80/050212-10$02.00/O Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

skin and can only be demonstrated in mice well after egg laying by the primary infection has commenced. A more effective phase of attrition occurs at a later stage of parasite migration, between Day 6 and 14 after challenge when the schistosomes have left the lungs. This “late phase” kills some 43% of the surviving challenge and can be demonstrated about the time the primary infection matures. These two separate phases of attrition might indicate two distinct mechanisms of immunity (Smithers and Gammage 1980). A recent encouraging advance in the vaccination of animals against schistosome infection with irradiated live organisms has

Schistosoma

mansoni:

ATTRITION

been the discovery that highly irradiated cercariae induce an immunity comparable to or even better than cercariae irradiated at lower levels (Hsii et al. 1%9; Minard et al. 1978a; Bickle et al. 1979a). Few highly irradiated cercariae are able to migrate out from the skin and it was of interest to us to determine whether immunity induced in this way, i.e., in the absence of mature adult worms and oviposition, was similar in mechanism to that induced during a chronic infection. We have therefore examined mice immunized with highly irradiated cercariae to determine whether the attrition of a challenge infection corresponds to the two phases of attrition seen in chronically infected mice. We chose as our experimental system mice immunized with 400 cercariae irradiated at 20 kR, since previous work by others (Bickle et al. 1979a) has shown that cercariae irradiated at 20 kR, provide the best stimulation of resistance and that using this dosage maximal resistance is induced by 4 to 6 weeks. MATERIALS

AND METHODS

Parasites and host. A Puerto Rican strain mansoni maintained in Biomphalaria glabrata was used in all

of Schistosoma

experiments. Female inbred CBAKa mice weighing 18-25 g were the experimental hosts. Irradiation of cercariae. Cercariae were concentrated in dechlorinated tap water to approximately 400/ml in a loo-ml glass beaker and exposed to 20 kR of y-irradiation from a cobaltt60 source at a rate of approximately 10,000 rad/min. Irradiated cercariae were used for infection within an hour of irradiation and within 3 hr of shedding from the snails. Infections. Mice were exposed to approximately 400 irradiated cercariae and challenged with 125- 158 normal cercariae percutaneously by the “ring” method (Smithers and Terry 1965). Briefly, each mouse was anaesthetized with Sagatal

IN IMMUNIZED

MICE

213

(pentobarbitone sodium, May & Baker Ltd, Dagenham) and the abdomen carefully shaved with animal clippers. A heavy metal ring with an internal diameter of 13 mm was placed on the shaved area and 0.4 to 1 ml of cercarial suspension pipetted into the ring. While exposing a batch of mice, six samples of the cercarial suspension were put aside and counted later in order to estimate the number of cercariae used for the challenge. Ten minutes were allowed for cercarial penetration, after which time less than 1% of the cercariae remained in the suspension. For intramuscular injections of schistosomula, irradiated cercariae were transformed mechanically into schistosomula (see Methods) and then injected intramuscularly into the hamstring muscle of the hind leg in volumes of 0.1 ml using a 25gauge needle. For challenge infections via the ear, holes 10 mm in diameter and 13 mm deep were drilled in a solid block of 25-mm-thick Perspex (Plexiglas). These conveniently held 0.4-0.5 ml of cercarial suspension. Mice were anaesthetized with Sagatal and laid on the Perspex block so that threequarters of one ear pinna was immersed in the cercarial suspension. Twenty minutes were allowed for cercarial penetration; approximately 20% of the cercariae failed to penetrate by this method of infection. Recovery of schistosomula from the skin.

Mice were killed individually by exposure to chloroform. An area of skin larger than that which had been exposed to cercariae (about 20 x 16 mm), but including the site of exposure, was removed with fine pointed scissors. The excised site was rinsed in RPM1 1640 pH 7.2, containing 20 mM Hepes, 100 units penicillin/ml and 100 pg streptomycin/ml, placed into a dry universal container, and cut into approximately 20 pieces. Eighteen mililiters of RPM1 1640 as described above, but also containing 0.05% collagenase (Sigma Chemical Co., St. Louis, MO.), 5% foetal calf serum (Flow Laboratories, Irvine,

214

MILLER

AND

Scotland), and 180 units of heparin was added to the container which was then incubated overnight. After incubation, the contents of each bottle were filtered through a stainless-steel sieve (36/in.2 mesh size) into a 20-ml conical-bottom plastic universal container. The filtrate was centrifuged at 1000 rpm for 3 min. The supernatant was removed to 0.5 ml and the sediment transferred into an Integrid tissue culture dish (60 x 15 mm with 2-mm grid, Falcon Plastics, Oxnard, Calif.) and the schistosomula counted at a magnification of x80. The recovered schistosomula were motile and appeared to be in good condition; dead schistosomula, i.e., flattened, nonmotile, opaque forms, were seen only rarely.

SMITHERS

system essentially as described by Doenhoff et al. (1978). Each mouse was perfused with 20 ml of citrate saline introduced into the left ventricle from a syringe; the worms were collected as they emerged with the perfusate from the opened hepatic portal vein into a conical-bottom plastic universal container. When perfusion was completed, about 100 mg of saponin was added to lyse the red blood cells and the perfusate was then centrifuged at 1500 rpm for 60 sec. Following centrifugation the sediment was transferred to an Intergrid dish for counting. Perfusates from mice challenged 21 or 14 days previously were counted at a magnification of x80; those from mice challenged 28 or 42 days previously, were counted at a magnification of x40. Preparation

of schistosomula. Cercariae lungs. The technique was modified from the shed within 2 hr from snails were transoriginal method of Sher et al. (1974) by in- formed artificially into schistosomula after creasing the incubation time and using a the method of Ramalho-Pinto et al. (1974). Hepes-buffered medium containing foetal Analysis of results. Student’s t test was calf serum. Following the removal of the used to analyse the statistical significance skin site of exposure, the thorax of the of differences between mean worm recovmouse was opened and the lungs were re- eries from control mice and those from immoved. After rinsing in RPM1 1640 con- munized mice. Where the mean worm retaining 20 mZt4 Hepes and antibiotics, the covery from the immunized mice (Z) was lungs were transferred to a dry universal less than the mean worm recovery from container and finely minced with sharp- control mice (C), the difference is expointed scissors. Eighteen milliliters of pressed as a percentage of the mean of the RPM1 1640 as described above but also control mice and represents the percentage containing 5% foetal calf serum and 180 immunity, i.e., units heparin was added to the lung tissue, c-7 percentage immunity =c x 100. and the container was incubated overnight (18-20 hr) at 37 C. After incubation, the contents of each RESULTS bottle was filtered, centrifuged, and the The Recovery of a Challenge Infection schistosomula were counted as described from Mice Immunized with Irradiated for the skin recovery procedure. To facili(20 kR) Cercariae, Compared with the tate visualisation of the schistosomula a Recovery of a Similar Infection from pinch of saponin was added to the sediment Control Mice to lyse the red blood cells just before counting. New or improved techniques of recovRecovery of schistosomes from the he- ering a cercarial infection from mice at patic portal system. Schistosoma mansoni various intervals after exposure (Smithers were recovered from the hepatic portal and Gammage 1980) have shown that in Recovery

of schistosomula

from

the

Schistosoma

mansoni:

ATTRITION

previously uninfected mice the numbers of Schistosoma mansoni recovered from the skin on the second day of infection, or from the skin and lungs on Days 3, 4, and 5, or from the liver on Days 14 and 21, do not differ significantly from the number recovered from the liver 4 or 5 weeks after infec-

c2i

c3i

c4i

c51 days

IN IMMUNIZED

215

MICE

tion when maximum recovery of parasites would be expected. The assumption has been made therefore that the recovery procedures used on Days 2-5 and 14 and 21 are effectively 100% efficient and can be used to trace the survival and attrition of a cercarial infection in mice.

C i

C i

6

after

28

C I

42

challenge

FIG. 1. Recovery of a challenge infection of Schisrosoma mansoni from the skin, lungs, and liver of control (c) and immunized (i) mice at different times after the challenge. The number of cercariae used for each challenge is shown for each experiment. Each histogram represents the mean recovery from a group of 6 mice. The values above the histograms give the percentage reduction in the recovery from the immunized group compared with the control group [c - ?)/c x 100; i.e., the percentage immunity. The significance of these values is shown by dots: : : P < 0.001; . . . P < 0.01; . . P < 0.02; . P < 0.0s; no dots = NS. The bars represent the SD.

MILLER

216

AND SMITHERS

sure. The results from the four largest experiments are expressed as histograms in Fig. 1 and the percentage reductions of the recoveries from the immunized mice compared with the control animals (i.e., percentage immunity) are given in Table I for all experiments. The recoveries from the control mice showed the expected consistency within each experiment and varied between 25 and 40% of the cercarial exposure. A loss of between 60 and 75% of the infection had already occurred by Day 2 and there was no

In this study we have used these recovery techniques to compare the survival of a challenge infection in mice immunized with highly irradiated cercariae with the survival of a similar infection in control animals. Mice were exposed percutaneously on the abdomen to 400 cercariae given 20 kR of 6oCo irradiation and challenged 4 or 5 weeks later, together with matched uninfected controls, at the same site with 127-158 normal cercariae. Recoveries of the challenge infection were made from groups of six mice at various times after the expo-

Percentage Immunity to Schistosoma

mansoni

TABLE I in Mice Immunized with Highly Irradiated (20 kR) cercariae” Experiment No.

Days after challenge

1

2

47****

2 3 4 5 6 14 21 28 42

15 4 55****

41*** 19 4t3**** 52*** 44*** 73****

go****

65****

72****

f3@***

3

4 41* 1 59**** 4***** 41*** go****

6

7

37***

8

57****

9

40***

35***

fj,**** (j)jC*** 76****

70*** Experiment

10

5 44****

11

43** 45**** *o**** 74**** 59**** g7**** go****

43*** 44****

*3****

64****

12

13

47***

,j@%*** 45**

72****

22***

14

15

16

17

57***

53****

42***

32***

51****

19”

56**** 63****

77****

(j,****

No.

77****

g,****

*2****

73****

Xb 42

44*

77**** 91****

18

60***

51 59 76 78

a Mice were immunized 4 to 6 weeks previously with 400 cercariae which had been given 20 kR @‘Co irradiation. Challenge infections of 120- 150 cercariae were recovered from the skin (Days l-2), skin and lungs (Days 3-6), or liver (Days 14, 21, 28,42). Percentage immunity is the percentage reduction in the mean recovery from groups of 6 immunized mice (I) compared with the recovery from groups of 6 control mice (C). Percentage immunity = [(C - 1)/C] X 100. * Arithmetic mean. c Values without asterisks are not significant. *P ** P ***p ****p

< 0.05. < 0.02. < 0.01. < 0.001.

Schistosoma

mansoni:

ATTRITION

evidence for any additional loss thereafter (Fig. 1). These results are in agreement with those of Smithers and Gammage (1980). As previously noted by these authors an assay of the surviving infection on Day 6 (from skin and lungs) cannot be relied upon to give maximum recovery of schistosomula, as some parasites may already begin to migrate out from the lungs on that day. When recoveries from the immunized mice were compared with the recoveries from the control animals, there was a clear indication that in the immune host a marked attrition of parasites had occurred as early as Day 2 after challenge. The average percentage immunity Tom 16 experiments on Day 2 was 42. A further attrition of some 13% was evident by Day 4 or 5 and recoveries from the liver on Days 14 and 21 indicated an overall immunity of 76-78% (Table I). These results indicate that the major attrition of the challenge infection had occurred by Day 4. The marked differences in recoveries from the skin, together with the failure of the vast majority of parasites to reach the lungs in immune animals, suggests that the major attrition occurred in the skin. It is not possible to say where the remaining parasites are killed as our assay techniques recovered only live worms. However, the additional attrition on Days 4 to 5 probably reflects a continuing trickle of parasite deaths from the effects of immunity operating in the skin. These deaths would take place in the skin or lungs. It is more difficult to predict the site of parasite attrition after Day 5; possibly this is merely a continuation of the trickle of deaths or there may be a situation similar to that which occurs in chronically infected mice where an effector mechanism distinct from the skin phase is operating after the parasites have left the lungs (Smithers and Gammage 1980). The Effect of the Route of Challenge on rthe Development of Immunity To determine whether the immunity which developed following exposure of

IN

IMMUNIZED

MICE

217

mice to highly irradiated cercariae was the result of local phenomena occurring at the site of cercarial penetration, mice immunized via the abdominal skin were challenged 4 weeks later at the same site or via the skin of the ear, with 140 cercariae. The challenge infection was recovered from groups of six mice from the skin on Day 2, from the skin and lungs on Day 4, and from the liver on Day 14. The results are shown in Fig. 2. Approximately one-third less of the challenge infection was recovered from control mice challenged via the ear when compared with recoveries from control mice challenged via the abdominal skin. Despite this difference, mice immunized in the abdominal skin demonstrated a similar pattern and level of resistance to the challenge infection whether challenged in the abdominal skin, or in the ear. These results demonstrate that local phenomena occurring at the site of immunization cannot account for the observed attrition of challenge infections in mice immunized with irradiated cercariae. The Effect of the Route of Zmmunization the Development of Immunity

on

Exposure of mice percutaneously to highly irradiated S. mansoni cercariae induced a high level of immunity within the skin. Two experiments were performed to determine whether sensitization of the skin by irradiated cercariae was a necessary prerequisite for immunity to be effective in the skin. Mice were immunized percutaneously with 400 irradiated cercariae or intramuscularly with 400 schistosomula which had been mechanically transformed from the same pool of irradiated cercariae. Four weeks later, groups of immunized mice and matched uninfected controls were challenged percutaneously with normal cercariae. The recovery of the challenge infections were assayed on Days 2, 4, and 21. The results are expressed in Fig. 3. Mice immunized percutaneously with ir-

218

challange-

MILLER

ab3

ear

ah3

2

ear

5 days

after

am

AND

14 ear

challenge

FIG. 2. Mice were immunized percutaneously in the abdomen with irradiated cercariae of Schisrosoma munsoni and 4 weeks later, together with groups of control mice, challenged with cercariae either in the abdomen (ahd) or in the ear (ear). Recovery of the challenge infection was made from the skin, lungs, and liver at different times after the challenge. For each pair of histograms the left-hand column represents the challenge control mice and the right-hand column the immunized mice. Each histogram represents the mean recovery from a group of 6 mice. The values, key, and significance of the values are expressed as in Fig. 1.

radiated cercariae developed the expected high level of resistance to the challenge infection. The pattern of parasite attrition was similar to that already described, with

2

4 days after challenge

21

FIG. 3. Mice were immunized via the abdominal skin (S) with irradiated cercariae or intramuscularly (M) with irradiated schistosomula of Schisrosoma mansoni and 4 weeks later, together with a group of control mice, challenged with cercariae. Recovery of the challenge infection was made from the skin, lungs, and liver at different times after the challenge. Each histogram represents the mean recovery from a group of 6 mice. The key, values, and significance of the values are expressed as in Fig. 1.

SMITHERS

immunity reaching 82-91% by Day 21. In contrast, immunization intramuscularly with irradiated schistosomula induced a level of immunity (46-47%) which was only half that seen in mice immunized percutaneously. In addition, the marked parasite attrition seen at Day 2 in the skin of mice immunized percutaneously was absent from mice immunized intramuscularly. These results demonstrate that immunization intramuscularly with irradiated schistosomula induces a much lower level of resistance than percutaneous challenge and suggests that penetration and perhaps the persistence in the skin of irradiated cercariae are necessary for the induction of a high level of skin immunity. The Effect of the Number of Irradiated Cercariae in the Immunizing Dose on the Development of Immunity To determine whether large numbers of irradiated cercariae were required for the induction of a high level of immunity, mice were immunized with various doses of irradiated cercariae. Groups of mice were immunized percutaneously with 50, 100, 200, or 400 irradiated cercariae; 4 weeks later the immunized mice and matched uninfected control mice were challenged with 138 cercariae. Challenge infections were recovered on Days 2,4, and 21. The results are shown in Table II. Mice immunized percutaneously with 400 irradiated cercariae developed an expected high level of immunity of 81%. There was only a slight decrease in the levels of immunity developed by mice immunized with 100 or 200 cercariae. In the mice which had only received 50 immunizing cercariae, the levels of immunity was only about half that seen in the other groups. This lower level of immunity however had already been attained by Day 2 and unlike the groups immunized with larger numbers of cercariae, there was no additional attrition of parasites beyond the skin stage. Clearly in this experiment, an immunizing dose of only 50 highly irradiated cercariae had induced a

~ChiSfoSO~U

mUnSOni:

ATTRITION

TABLE II Percentage Immunity to Schisrosoma mansoni in Mice Immunized with Varying Numbers of Irradiated (20 kR) Cercariae” Days after challenge No. of cercariae SO 100 200 400

2

4

39*** 49*** 49*** 57***

42*** 46*** 47***

56****

21 40* 71**** 69**** g1****

(1Mice were immunized 4 weeks previously with varying numbers of cercariae which had been given 20 kRBOCo irradiation. Recoveries of a challenge infection of 138 cercariae were made from the skin (Day 2), skin and lungs (Day 4), or liver (Day 21). Percentage immunity is the percentage reduction of the mean recovery from groups of six immunized mice compared with the mean recovery from groups of six control mice. Percentage immunity = [1; - 1)/C] X 100. * P < 0.05. ***p < 0.01. ****p < 0.001.

skin immunity almost equivalent to that obtained with larger immunizing doses, although the final overall immunity remained lower. of Irradiated from Normal Mice

Recovery

(20 kR) Cercariae

In order to ascertain the survival and migratory behaviour of cercariae given 20 kR 6oCo irradiation, recoveries of an immunizing infection of 400 irradiated cercariae were made from the skin, lungs, and liver of control mice. This experiment also enabled us to compare the effects of our dose of irradiation on cercarial behaviour with the dosage used by other workers. Mice were exposed percutaneously to 400 irradiated cercariae. Infections were recovered from the skin (Days l- 14), the lungs (Days 3 - 14), and from the liver (Day 21) and the percentage survival was calculated. The results of a representative experiment are shown in Table III. Approximately 12% of the infection was recovered from the skin on Days 1 and 2; 8-10% of the schistosomula were still recoverable

IN IMMUNIZED

219

MICE

TABLE III Recovery of Irradiated (20 kR) Cercariae of Schistosoma mansoni from Normal Mice” Percentage recovery*

Days after challenge

Skin

Lung

1 2 3 4 5 6 7 8 10 14 21

11.5 13.9 8.2 8.8 5.7 2.9 1.7 3.1 1.0 1.2 -

0.5 1.5 2.0 1.8 1.6 2.3 1.1 0.1 -

Liver

Total

0.1

11.5 13.9 8.7 10.3 7.7 4.7 3.3 5.4 2.1 1.3 0.1

a Mice were exposed to 400 cercariae which had been given 20 kR of Wo irradiation. b Percentage recovery = (mean recovery/No. of cercariae) X 100. Each value represents the mean from groups of six mice.

from this site on Days 4 and 5. Thereafter there was a gradual decline in the percentage of worms recovered from the skin with 1% of the infection still remaining at Day 14. Approximately 2% of the infection matured to the lung stage and only a small proportion, 0.1% of the infection, was recovered from the liver on Day 21. This pattern of survival and migration of cercariae irradiated with 20 kR is similar to the findings of Bickle et al. (1979a) in that the majority of attenuated organisms failed to leave the skin. As these authors point out, however, a similar pattern of migration and behaviour was observed by Minard et al. (1978b) using cercariae exposed to 56 kR. DISCUSSION

These results have demonstrated that 4-5 weeks after exposure of mice to highly irradiated (20 kR) cercariae of Schistosoma mansoni, there was a reduction in the survival of a challenge infection of approximately 77% compared to control mice. This substantial level of immunity to reinfection

220

MILLER

AND

is equivalent to that seen in chronically infected mice lo- 12 weeks after exposure to 20 cercariae (Smithers and Gammage 1980). Similar levels of resistance in mice immunized with highly irradiated cercariae have been observed by Minard et al. (1978a) and Bickle et al. (1979b). Although the level of resistance developed in mice after exposure to highly irradiated cercariae is similar to that developed during a chronic infection, there is a marked contrast in the pattern of attrition of the challenge infection following the different methods of inducing resistance. In mice immunized with highly irradiated cercariae, the majority of parasite attrition occurs within the skin and in the first 4 days after challenge. The death of the remaining parasites appeared to take place as a trickle, but it was not possible to ascertain from our recovery techniques where these deaths had occurred. In mice which have been recently infected with 20 cercariae, the attrition of the challenge infection takes place between Days 6 and 14, after the parasites have migrated from the lungs and are either en route to the liver or have just arrived in that organ. In mice infected for 10 weeks or more, there may be an additional early phase of attrition in the skin, possibly equivalent to that seen in mice immunized with highly irradiated cercariae, but in the chronically infected host this phase is never the major contribution to parasite death (Smithers and Gammage 1980). The two distinct phases of parasite attrition seen in chronically infected mice have led us to suggest that there may be two separate mechanisms of immunity of schistosome reinfection (Smithers and Gammage 1980). The predominance of the skin phase of attrition in mice immunized with highly irradiated cercariae and the absence, or at least the minor role of the postlung phase, would add further support to this suggestion. We have confirmed the earlier findings of Bickle et al. (1979b) that the intramuscular

SMITHERS

route of immunization (by injecting highly irradiated schistosomula) is approximately only half as effective in stimulating resistance as the percutaneous route. In addition, the marked attrition of the challenge organisms which occurred by Day 2 in the skin of the percutaneously immunized mice was absent from the mice immunized intramuscularly. The absence of a strong skin immunity when immunization bypassed the skin suggests that penetration and perhaps persistence of live attenuated organisms in the skin are necessary prerequisites for the induction of skin immunity. Ten to twelve percent of our irradiated cercariae persisted in the skin for 4-5 days and small numbers of live organisms were still present on Day 14. Furthermore, a dose of 50 highly irradiated cercariae was more efficient at inducing skin immunity than an intramuscular injection of 400 irradiated schistosomula, although the overall level of resistance was similar. We therefore suggest that penetration and/or persistence of attenuated schistosomula in the skin may be a crucial factor in the induction of high levels of skin immunity with highly irradiated cercariae. The observation, however, that skin immunity eventually develops in chronically infected mice indicates that this mechanism can be induced in other ways. In this paper we have demonstrated that mice immunized with highly irradiated cercariae develop a strong immune response which is largely effective in the skin. These results extend the earlier findings of Hsu et al. (1963) who observed in monkeys and mice that multiple infections with irradiated cercariae could shift the site of schistosomula destruction from the liver to the lungs and skin. Immunity induced in this way differs from the resistance developed during a chronic infection where the majority of parasite attrition occurs after the lung phase of migration. Skin immunity, however, may prove to be the most convenient mechanism to induce in vaccine development, whether live attenuated organisms, or antigenic extracts of the parasites are

,!khistosomU

WMZnsotk

ATTRITION

used. The immunization of mice with highly irradiated Schistosoma mansoni cercariae and the assay of immunity by skin or skin and lung recovery techniques provide a suitable model for further study of this immune mechanism.

The authors wish to thank K. Gammage and Elaine Holder for their excellent technical assistance. Dr. Kathleen Miller was funded by Grant AI 05759 from the U.S. National Institutes of Health.

REFERENCES Q. D., DOBINSON, T., AND JAMES, E. R. 1979a. The effects of gamma-irradiation on migration and survival of Schistosoma mansoni schistosomula in mice. Parasitology 79, 223-230. BICKLE, Q. D., TAYLOR, M. G., D~ENHOFF, M. J., AND NELSON, G. S. 1979b. Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schisrosoma mansoni. Parasitol-

BICKLE,

ogy 79, 209-222. DOENHOFF, M. J., BICKLE, J., AND MCGREGOR, A.

acquisition in the reinfection fusion of

soni

Journal

of

Q. D., LONG, E., BAIN, 1978. Factors affecting the of resistance against Schistosoma manmouse. I. Demonstration of resistance to using a model system that involves permice within three weeks of challenge. Helminthology 52, 173 - 186.

HsO, H. F., DAVIS, J. R., HsO, S. Y. LI, AND OsBORNE, J. E. 1963. Histopathology in albino mice and rhesus monkeys infected with irradiated cer-

221

MICE

cariae of Schistosoma japonicum. Zeitschrift Tropenmedizin und Parasitologie 14, 240-261. Hso, S. Y. LI, HsO, H. F., AND OSBORNE, J. W.

fur

1969. Immunization of rhesus monkeys against schistosome infection by cercariae exposed to high doses of X-irradiation. Proceedings of the Society for Experimental Biology and Medicine 136, 727-731.

MINARD, NIER,

ACKNOWLEDGMENTS

IN IMMUNIZED

P., DEAN,

D. A.,

JACOBSEN,

R. H.,

VAN-

W. E., AND MURRELL, K. D. 1978a. Immunization of mice with cobalt-~ irradiated Schistosoma mansoni cercariae. American Journal of Tropical

Medicine and Hygiene 27, 76-86. MINARD, P., DEAN, D. A., VANNIER, W. E., AND MURRELL, K. D. 1978b. Effect of immunization of migration of Schistosoma mansoni through lungs. American Journal of Tropical Medicine and Hygiene 27, 87-93. RAMALHO-PINTO, F. J., GAZZINELLI, G., HOWELLS, R. E., MOTA-SANTOS, T. A., FIGUEIREDO, E. A., AND PELLEGRINO, J. 1974. Schistosoma mansoni

defined system for stepwise transformation of cercariae to schistosomula in vitro. Experimental Parasitology 36, 360-372. SHER, A., MACKENZIE, P.

E., AND SMITHERS, S. R. 1974. Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the laboratory mouse. Journal of Infectious Diseases 130, 626-633. SMITHERS, S. R., AND GAMMAGE, K. 1980. Recovery of Schistosoma mansoni from the skin, lungs and hepatic portal system of naive mice and mice previously exposed to S. mansoni: Evidence for two phases of parasite attrition in immune mice. Parasitology SMITHERS,

80, 289-300.

S. R., AND TERRY, R. J. 1965. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms.

Parasitology

55, 695 -700.