Studies on schistosomiasis. XVII. Non-reciprocal acquired resistance between Schistosoma mansoni and Schistosomatium douthitti in mice

EXPERIMENTAL

PARASITOLOGY

11,

133-140

(1961)

Studies on Schistosomiasis. XVII. Non-Reciprocal Resistance between Schistosoma mansoni and tium douthitti in Mice* George

College

Acquired Schistosoma-

W. Hunter, III,l Clarence J. Weinmann,2 Robert G. Hoffmann of Medicine, (Submitted

University for

of Florida,

publication,

Gainesville,

1 March

and

Florida

1961)

reactions between Schistosoma mansoni and Attempts to study “cross immunity” Schistosomatium douthitti revealed a non-reciprocal, partial acquired resistance. Mice which were “immunized” by 5 exposures of 10 cercariae each to S. douthitti and challenged with 50 S. mansoni cercariae did not show any reduction in the number of S. nzansoni adults recovered when compared with the challenge control. However, the with similar numbers of S. mansoni reciprocal cross in which the mice were “immunized” cercariae and challenged with S. douthitti, showed a highly significant reduction in s. douthitti in comparison with the challenge control. The homologous crosses showed little evidence of “immunity” upon challenge.

experiments. Only male mice, initially about 6 weeks of age, were employed. Mice were “immunized” by five exposures to 10 cercariae of either S. mansoni or S. douthitti at intervals of lo-14 days. Cercariae for each exposure were obtained from pools of 14-34 Australorbis glabratus shedding S. mansoni and 8-16 Lymnaea palustris positive for S. douthitti4 Challenge infections consisted of exposure to 50 cercariae of either the homologous or heterologous speciesabout 4 to 5 weeks after the final “immunizing” exposure. Controls for the “immunizing” series of infections and challenge exposures were established. All animals (except a few immunizing controls) were sacrificed about 6 MATERIALS AND METHODS weeksafter the final exposureto cercariae and Swiss mice of the Webster strain from Cartheir worm burdens determined. The experiworth Farms (CFW) were utilized in these mental designand scheduleof the three repli* This investigation was supported by Grant Ecate experiments are illustrated in Fig. 1. 1893 from the National Institute of Allergy and Mice were exposed to cercariae in two Infectious Diseases, National Institutes of Health, different ways as follows: In the first experi-

The phenomenon of cross immunity between related parasites is well known for diverse pathogens.Evidence suggestinga nonreciprocal cross immunity was obtained in experiments on the tissue responseof small mammals to penetration by Schistosoma japonicum and Gigantobilharzia sturniae (Hunter et al., 1949, 1956). These observations prompted further experimentation with other schistosomespecies.The present report concerns a study of cross resistance in mice initially infected with either Schistosomamansoni or Schistosomatium douthitti when challenged by the homologous or heterologous parasite.

Public Health Service. 1 Department of Microbiology, College of Meditine, University of Florida, Gainesville. 2 Department of Biology, Rice University, Houston, Texas. 3 J. Hillis Miller Health Center, University of Florida, Gainesville.

4 The snails for these experiments were from Dr. L. S. Ritchie’s A. glabratus colony in Puerto Rico and the L. palzdstris were originally obtained from the laboratories of Dr. C. G. Goodchild of Emory University and Dr. R. B. Short of Florida State University. 133

134

HUNTER,

SCHISTOSOMA SMI

SMXSM

WEINMANN,

AND

MANSONI

HOFFMANN

SCHISTOSOMATIUM

SMXSD

94 a 4

SDXSM

ABOUT DAYS

SDI

I

“IMMUNIZIfylG” EXPOSURES IO-14

SDXSD

DOUTHITTI

+ APART

0 ”

; SDC

SMC @ TO

50

AUTOPSIED IN ABOUT 6 WEEKS

FIG. 1. Explanation of abbreviations immunizing control; SM = S. mansoni; hitti; SD1 = S. douthitti immunizing

used in protocols of the several experiments: SMI = S. mansoni SMC = S. mansoni challenge control; SD = Schistosomatium doutcontrol; SDC = S. douthitti challenge control.

ment, each animal was taped onto a plastic infection board. A known number of cercariae, counted in drops of water on microscope slides, were carefully washed into the wells after which the slides were checked for cercariae. In the next two experiments the mice were allowed to wade in tepid spring water for 30 minutes prior to exposure to encourage defecation and urination. They were then placed individually in finger bowls (diameter 11.5 cm) into which a known number of cercariae had been counted. Sufficient tepid spring water was added to immerse the feet and abdominal surfaces. The abdominal hair of all mice was clipped prior to exposure to cercariae. The mice were exposed for 30 minutes. A modification of the perfusion technique of Radke et al. (1957) was utilized to recover the parasites. Before sacrificing the mice an anticoagulant and chlorobutanol was injected I.P.; this greatly facilitated perfusion

(Hunter, 1960). Following perfusion, the mesenteries,liver and other organs were compressed between two plastic plates and examined under a stereoscopic microscope for worms. In addition the liver was routinely teased apart after compression; occasionally the lungs and spleen were also examined in this manner. The recovered parasites were relaxed in spring water and the number, sex, species, and degreeof maturation were recorded. The criterion for the development of host resistance was the number of parasites and their maturity that developed after a challenge infection compared with the number and maturity of worms recovered from control animals. The rationale of the experiment was that if no resistance developed after a series of light infections, the mean number of parasites recovered from test mice should approximate the mean burden of the immunizing controls plus the challenge controls.

STUDIES

-

SCHISTOSOMIASIS.

SD x SM coruuDmy

SMXSD (CO~;lCE&G FIG.

ON

2.

Per cent of worms

recovered

SDXSD

from

In heterologous challenges, a direct comparison between the challenge and challenge control mice was made. In each experiment, the test and control mice were exposed at the same time to cercariae from the same pool. RESULTS

Experimental results are presented in Table I. The results of the homologous and heterologous infection series combining all three replicates are illustrated in Fig. 2 which is a comparison of the per cent of worms recovered from the test and challenge control mice. It may be seen that the mice initially exposed to S. mansoni and given the homologous challenge harbored essentially the same number of worms as the immunizing and challenge controls combined (Table I). The differences in mean worm burden were not statistically significant, indicating that these animals had not acquired a significant

various

XVII

soItsDc

infections

in three

experiments.

degree of resistance to reinfection (Fig. 2). The “t” values shown in Table I were computed in two somewhat different ways. Where a heterologous cross was made the challenge parasites could be identified and the ordinary “t” test computed. Where homologous crosses were involved, however, the experimental group of mice should be compared with the sums of the averages of the immunizing and challenge control groups. A further complication from the statistical viewpoint, was that the variability within the two control groups was small compared with the treated group. As a conservative approach to a probability statement, we took the difference between the treated group mean and the sum of the means of the control groups as the numerator of ‘(t”. The denominator of “t” was simply the standard deviation of the treated group mean. Only two of the experiments included a

SM x SM

1

2 3

1

2 3

1

2

the

Data

SD SD SD

SM SM SM

SD SD SD

SM SM SM

SD SD

SM SM SM

Challenged by

Experimental

Secured

SD SD

SM SM SM

Immunized by

Raw

in Cross

(SW

(8-17) (5-19)

11.1 12.8

(O-10) (9-23) (2-12)

(20-43)

(17-36)

(2-18) (6-31) (m-23)

(I-13)

(SW

(SD)

(SD)

(O-19) (2-14) (9-17)

12.33

<.OOl <.OOl

<.OOl

.l

(l-6)

4.9

7.68 6.64

.2 .3

(6-21)

<.ool”

.2

.2

.3 .7

P valuea

Schistosoma

(6-11)

(SD)

with

(Range)

I

8.4

4.5

(SW

TABLE Experiments

Mean of Experimental Total (Range)

Immunity

5.78

14.8 13.4 16.57

28.6

23.6

16.3

8.28 14.8

Total

infections

Three

(SM)

7.4 10.9

5.9

12.75

13.2 6.87

6.87

13.2

10.9

5.9 7.4

(SMU

(SD0

(=‘I)

(SMI)

(4-11) (8-16)

(z-9)

(8-22)

(6-20) (3-9)

(3-9)

(6-20)

(8-16)

(4-11)

(z-9)

Mean of immunizing Control (Range)

mansoni

and

12.72 23.44

16.08

7.13

3.04 6.8

12.72

16.1

7.13

3.15 6.8

@DC)

(SMC)

WC)

(SMC)

(l&15) (17-31)

(9-29)

(3-13)

C&5) (4-13)

(m-15)

(9-29)

(3-13)

(o-5) (4-13)

Mean of challenge Control (Range)

Controls

Schistosomatium

of caltotalsc

(SD)

-

-

-

-

--

19.6

29.3

18.0

9.05 14.2

Mean culated

douthitti

a The P value is the probability that the mean of the experimental group and its control(s) could have come from the same population. b See “Discussion” in text for explanation. c The number in the “calculated total” is the sum of the means of the immunizing plus the challenge controls. The following abbreviations are used: SM = S. mansoni; SMI = Immunizing control for S. mansoni; SMC = Challenge control for S. mansoni; SD = S. douthitti; SD1 = Immunizing control for S. douthitti; SDC = Challenge control for S. douthitti.

SM x SD

SD x SM

SD x SD

Schedule

2 3

Expt. No.

1

of

Summary

8 s z

g

$ 3

2

$ E

z P

STUDIES

ON

SCHISTOSOMIASIS.

homologous challenge with S. douthitti; the results were not in agreement. In Experiment 1 there was a small reduction in worm burden as indicated by the combined controls, but this was not statistically significant (Table I). However, in Experiment 2 the animals receiving S. douthitti in both the “immunizing” and challenge exposures harbored a significantly larger number of parasites than the sum of the immunizing plus the challenge controls. It should be noted here that these mice were quite debilitated at the time of challenge. In the heterologous challenge of mice by S. mansoni, it may be seen that the mean number of parasites of this species in both the test and challenge controls was comparable, indicating that prior infection with S. douthitti did not influence subsequent resistance to S. mansoni. Essentially similar results were obtained in all three of the replicates (Fig. 2). In contrast, the animals “immunized” by 5’. mansoni and then challenged by S. douthitti showed a marked reduction in the challenge worm burden when compared with the challenge control groups. The difference in mean challenge worm burden between test and control mice was highly significant in each of the three experiments (Table I). In none of the experiments was there any evidence of “stunting” or reduction in size of the recovered worms which could be interpreted as being due to the “immunizing” procedures. DISCUSSION

The development of acquired resistance to homologous challenge by S. mansoni and S. douthitti has been reported by a number of investigators. Generally a low grade “immunity” can be produced in experimental animals which is manifested by a reduction in worm burden or an inhibition of parasite development (Kagan and Lee, 1953). These criteria have indicated that animals, such as mice and hamsters, usually acquire some degree of resistance after repeated exposures to small numbers of cercariae, although results of various workers have not, been entirely consistent (Kagan, 1952; Olivier and Schneidermann, 19.53; Stirewalt, 1953;

XVII

137

Anonymous, 1954; Lin et al., 1954; Thompson, 1954; Gonnert, 1955). It was expected that a challenge by the homologous parasite following five “immunizing” exposures to 10 active cercariae each would result in a detectable reduction of worms from the challenge exposure. As already noted (vida supra) results recorded in the literature have not been consistent especially in regard to S. mansoni. In this experiment it is believed that the absence of any evidence of “immunity” or resistance, following challenge by the homologous parasite in S. mansoni infections, was due to the fact that only 4 to 5 weeks elapsed between the last “immunizing” and challenge exposures. Kagan and Lee (1953) have suggested that the presence of viable eggs is important in the production of resistance to challenge in the case of S. douthitti. If true it probably holds for the other schistosomes as well. It was felt at the time of the experiment that these 4 to 5 weeks provided sufficient time between “immunization” and challenge for the development of resistance. This view was supported by the presence of both lesions and viable eggs in the “immunizing controls” examined at the time of challenge. However, subsequent unpublished experiments by one of us (GWH) indicate that the interval to challenge is an important factor in the development of “immunity” or resistance to challenge by the homologous parasite. In this series of experiments the time interval may not have been sufficiently long. It is fairly well established, however, that mice “immunized” to S. douthitti and subsequently challenged with the homologous parasite will yield fewer worms in comparison with the controls (Kagan, 1952). This was the principle reason for our not running more replicates of this cross. As noted previously in the first experiment, the homologous cross (SD X SD) showed a marked reduction in worms recovered (Table I), but the second replicate did not show this same trend. This may be explained by the observation that, at the time of challenge, the test animals were obviously debilitated. Debilitation was believed due to some extraneous infection (viral or bacterial) which apparently re-

138

HUNTER,

WEINMANN,

sulted in increased susceptibility-a condition not evident in the controls. The consistent and marked effect of prior exposure to S. mansoni on subsequent infection with S. douthitti stands in contrast to the apparent absence of cross resistance in the reciprocal infections. Non-reciprocal cross immune responses have been reported for various parasites (Taliaferro and Taliaferro, 1945) and non-reciprocal cross reactions have been demonstrated with synthetic conjugated antigenic substances (Landsteiner, 1945). In general, cross reactions occur only between closely related antigenic substances and when cross immunity is observed in infection experiments, it is usually assumed that common and/or related antigenic materials are involved (presupposing adequate evidence that an antibody mechanism is important in the development of resistance). Furthermore, it has generally been observed that the strongest immunological response or reaction occurs with the homologous antigen (Culbertson et al., 1947). In a cross infection system which includes a marked heterologous interaction, one would expect a comparable homologous response if the interaction being measured were primarily or directly the consequence of immunological phenomona. The present observations do not seem explicable on this basis. Resistance to reinfection is undoubtedly the result of a complex series of responses, some specifically elicited by the parasite and others non-specifically stimulated. In schistosome infections, little is known regarding the role of antibody in the development of host resistance, but circumstantial evidence indicates that immunological events are involved (Kagan, 1958). However, non-specific factors may prove to be equally important. The phenomenon of non-specific interference is probably not confined to bacterial and viral systems and should be considered as a possibility in the present instance. Non-specific preparation, of the host’s defensive array seems to underlie resistance to superinfection with immunologically unrelated agents (Henderson, 1960) and it seems likely that this resistance would be operative even in the presence of an antibody response. But there is little information on which to base an

AND

HOFFMANN

appraisal of the relative importance of the various components of the host response to schistosome invasion. An increased cellular response is known to occur in the skin and in the lungs upon reinfection (Hunter et al., 1949, 1956; Kagan and Meranze, 1957; Stirewalt and Hackey, 1956; Lin and Sadun, 1959; Sadun and Lin, 1959) and there is evidence of worm destruction within the lungs under certain conditions (Magalhaes, 1959; Hewitt and Gill, 1960). To what extent these reactions are dependent upon specific elicitation is unknown. Studies are underway to elucidate the immunological relationships between these parasites and the host reaction to reinfection. The lack of reciprocity in the heterologous challenge infections suggests that there are differences between the species with regard to vulnerability to a host environment altered in some way as a consequence of previous infection. These differences could be primarily physiological in nature. There is some evidence that these parasites do differ in pathogenesis (Kagan and Meranze, 1957) and in temporal and spatial relations within the mouse host. Olivier (1952) observed that S. douthitti rapidly pass through the lungs of mice and reach the portal system in 4 to 7 days, while S. mansoni in the mouse host is retained in the lungs between the fourth and tenth days of infection before entering the portal system in numbers. It was also noted that there seemed to be differences in the rate of maturation within the portal system. It has generally been reported that S. mansoni is confined to the vascular system in its migrations in the host (Meleney and Moore, 1954), but S. douthitti has been observed in extra-alveolar positions in the mouse host (Kagan and Meranze, 1957) and El-Gindy (1951) has postulated an extravascular route of migration. These observations suggest the possibility that S. douthitti may encounter different host barriers as well as different temporal relations in the mouse host. There is some reason for considering the mouse as a better host (i.e., a more “normal” host) for S. douthitti than for S. mansoni (Olivier, 1952). If this is so, it may be said that the less well-adapted parasite (S. man-

STUDIES

ON

SCHISTOSOMIASIS.

soni) provoked in the mouse a response sufficient to affect the fate of S. douthitti when these subsequently invaded the host. But the better adapted S. douthitti on primary infection did not elicit a response that appreciably affected later invasions by S. mansoni. From our data, it must also be assumed that the host response elicited by either parasite was not sufficient to influence detectably reinfection with the homologous parasite. This would imply that S. douthitti is particularly sensitive or vulnerable to the host reactions elicited by S. mansoni. As suggested earlier it seems more likely that this rather anomalous situation is due to peculiarities or differences in the parasites themselves than to specific host dependent reactions.

139

XVII

G. W., III. 1960. The use of anticoagulants and chlorobutanol for the recovery of adult schistosomes from mice. Journal of Parasitology

HUNTER,

46,

206.

I. G. 1952. Acquired immunity in mice infected with Schistosomatium douthitti. Journal of Znfectious Diseases 91, 147-158. K~GAN, I. G. 1958. Contributions to the immunology and serology of schistosomiasis. Rice Institute Pamphlet 45, 151-183. KAGAN, I. G., AND LEE, C. L. 1953. Duration of acquired immunity of Schistosomatium douthitti infections in mice following treatment. Journal of Infectious Diseases 92, 52-57. KAGAN, I. G., AND MERANZE, D. R. 1957. The histopathology of the liver in mice experimentally infected with Schistosomatium douthitti. Journal of Infectious Diseases 100, 32-39.

KAGAN,

K. 1945. The specificity reactions. 2nd ed. Harvard Univ. bridge, Mass.

LANDSTEINER, REFERENCES

1954. 406th Medical General Laboratory Professional Report 1954. U.S. Army Forces, Far East. pp. 289. Tokyo, Japan.

ANONYMOUS.

H. T., ROSE, H. M., AND OLIVER-G• N1947. Skin tests in schistosomiasis mansoni with antigen from heterologous worms (Pneumoneces; Planaria). Journal of Infectious Diseases 80, 2 18-2 2 I.

CULBERTSON,

J.

ZALEz,

M. S. 1951. Post-cercarial development of Schistosomatium douthitti (Cort, 1914) Price 1931 in mice, with special reference to the genital system. (Schistosomatidae-Trematoda). Journal of Morphology 89, 151.186.

EL-GINDY,

R. 1955. Schistosomiasis-Studien. II. Uber die Eibildung bei Schistosoma mansoni und das Schicksal der Eier im Wirtsorganismus. Zeitschrift fiir Tropenmedicin und Parasitenkunde

GBNNERT,

6,

33-52.

D. Bacteriological

HENDERSON,

W. 1960. Bacterial Reviews 24, 167-176.

interference.

EDNA. 1960. The “lung shift” of Schistosoma mansoni in mice following therapy with tartar emetic or Miracil D. American Journal of Tropical Medicine and Hygiene 9, 402-409. HUNTER, G. W., III, RITCHIE, L. S., TIGERTT, W. D., LIN, S., PAN, C., AND TANABE, H. 1949. Immunological studies. I. Experiments with bird and human schistosomes. Journal of Parasitology (Suppl.) 35, 28-29.

HEWITT,

R.

AND

GILL,

G. W., III, RITCHIE, L. S., LIN, S., PAN, C., H. 1956. Immunological studies. I. Experiments with bird and human schistosomes in small mammals. E:~perimentaZ Parasitology 5, 551-559.

HUNTER,

AND

TANABE,

LIN,

S.,

RITCHIE,

L.

S., AND

HUNTER,

of serological Press, CamG.

W.,

III.

1954. Acquired immunologic resistance against Schistosoma japonicum. Journal of Parasitology (Suppl.) 40, 42. LIN, S., AND SADUN, E. H. 1959. Studies on the host parasite relationships to Schistosoma japonicum. V. Reactions in the skin. lungs_ and liver of normal and immune animals following infection with Schistosoma japonicum. Journal of Parasitology 45, 549-559. F. A. 1959. Pulmonary lesions in mice experimentally infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 8, 527-535.

MAGALHAES,

H. E., AND MOORE, D. V. 1954. Observations on immunity to superinfection with Schistosoma mansoni and S. haematobium in monkeys. Experimental Parasitology 9, 128-139.

MELENEY,

L. 1952. .4 comparison of infections in mice with three species of schistosomes, Schistosoma mansoni, Schistosoma japonicum and Schistosomatium douthitti. American Journal of Hygiene

OLIVIER,

55,

22-35.

L., AND SCHNEIDERMANN, M. 1953. Acquired resistance to Schistosoma mansoni infection in laboratory animals. .4merican Journal of Tropical Medicine and Hygiene 2, 298-306.

OLIVIER,

M. G., BERRIOS-DURAN, L. A., AND MORAN, K. 1957. A perfusion procedure (PERF-O-SUCTION) for recovery of schistosome worms. Journal of Parasitology (Suppl.) 43, 26-27.

RADKE,

E. H., AND LIN, S. 1959. Studies on the host parasite relationships to Schistosoma japonicum. IV. Resistance acquired by infection, by vac-

SADUN,

HUNTER,

140

WEINMANN,

AND

HOFFMANN

cination and by the injection of immune serum, in monkeys, rabbits, and mice. Journal of Parasitology 46, 543-54s.

mansoni. I. Observed entry into skin of mouse, hamster, rat, monkey and man. Journal of Parasitology 42, 565-580.

STIREWALT, M. A. 1953. The influence of previous infection of mice with Schistosoma mansoni on a challenging infection with the homologous parasite. American Journal of Tropical Medicine and Hygiene 2, 867-882.

TALIAFERRO, W. H., AND TALIAFERRO, L. G. 1945. Immunological relationships of Plasmodium gallinaceum and Plasmodium lophurae. Journal of Infectious Diseases 77, 224-248.

STIREWALT, tration

M. A., AND HACKEY, J. R. 1956. Peneof host skin by cercariae of Schistosoma

THOMPSON, J. H., JR. 19.54. Host-parasite relationships of Schistosoma mansoni. Experimental Parasitology 3, 140-160.