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Trichinella spiralis and Strongyloides ratti: Immune interaction in adult rats
EXPERIMEMTAL
PARASITOLOGY
47, 65-72
(1979)
Trichinella spiralis and Sfrongyloides ratti: Immune Interaction in Adult Rats R. MOQBEL AND D. WAKELIN Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Bearsden Road, Bearsden, Glasgow, G61 1 QH, Scotland, United Kingdom (Accepted
for publication
I7 October
1978)
MOQBEL, R., AND WAKELIN, D. 1979. Trichinella spiralis and Strongyloides ratti: Immune interaction in adult rats. Experimenta Parasitology 47, 65-72. The consequences of prior and concurrent infection with two species of nematodes were studied in rats. Primary infection with Strongyloides ratti adversely affected the development of a secondary Trichinella spiralis infection. Both immediate and delayed challenge with T. spiralis, following the expulsion of the previous S. ratti infection, reduced the percentage of worm recovery of the former as well as their fecundities and lengths. It is suggested that nonspecific inflammation produced by one species, during the peak period of worm expulsion, was not responsible for the accelerated rate of expulsion of the other; instead a direct, specific cross-immunity was probably operative affecting the survival of the challenge species. The response elicited by previous experience of the intestinal phase was reciprocal, but there was evidence of an enhancing effect by the muscle larval stages of T. spiralis on S. ratti. Rats concurrently infected with both species expelled S. ratti more rapidly than T. spiralis. Possible mechanisms underlying the interaction between the two species are suggested and discussed. INDEX DESCRIPTORS: Trichinella spiralis; Strongyloides ratti; Cross-immunity; Concurrent infections; Immune expulsion; Intestinal inflammation; Immunosuppression; Rat; Nematode.
ever, in the majority of cases, the crossimmunity arises indirectly from the immune response, most commonly as a result of the intestinal “allergic” inflammation (reviewed by Larsh and Race 1975) evoked by one species acting nonspecifically against the other species present (Stewart 1953; Bruce and Wakelin 1977; Behnke et al. 1977). Direct and indirect cross-immunity can be differentiated when infection with the challenging species is given a long time after the initial infection, i.e., when the intestinal environment has returned to normal and the manifestations of the inflammatory response have subsided.
The phenomenon of “cross-immunity” between species of intestinal helminths has been widely studied because of its potential significance in the context of naturally occurring infections, both in man and domestic animals. In a number of species combinations, prior or concurrent infection with one species will adversely affect the establishment or survival of a second (reviewed by Kazacos 1975). It has often been assumed that this effect has a direct immunological basis, i.e., the species concerned share common antigens and the immune response evoked by one species operates directly against the other. How65
0014-4894/79/010065-08$02.00/O All
Copyright 0 1979 rights of reproduction
by
Academic Press, Inc. in any form reserved.
MOQBEL
AND WAKELIN
between T. spirulis nld S. rutti h rats, using an initial infection with one species as the source of sensitization against the other; (ii) the reciprocity of the response; (iii) the effect of the acute (intestinal phase) and chronic (muscle larval phase) stages of T. spiralis 011 the course of S. ratti infection; (iv) the course of concurrent infections with both species in rats. MATEHIALS
I
1”
DAYS AFTER
2”
INFECTION
FIG. 1. Schematic diagram of the course of primary and secondary infections with Trichinellu spidis (open bars) and Strongyloides ratti (closed bars) in adult rats (t) Day of secondary infection.
The two nematodes studied here evoke strong immunity in the rat host. Rats initially infected with Strongyloicles ratti or TrichineEZa spiralis manifest the phenomenon of spontaneous cure (Fig. 1) and subsequently exhibit strong resistance to reinfection (Moqbel and Denham 1977; Love et al. 1976; McCoy 1931). During primary and secondary infections with either species, resistance is reflected in changes in worm fecundity and morphology as well as in the position occupied in the small intestine (Moqbel and Ellis 1978; Love et al. 1976). Immunity to T. spiralis is associated with marked intestinal inflammation. S. ratti, on the other hand, induces a number of histopathological changes in the small intestine (Moqbel 1977), but the degree of inflammation is much less pronounced than in rats infected with T. spiralis. The experiments described in this report were designed to investigate the folIowing points: (i) th e nature of cross-reaction
AND METHODS
Some of the experiments described in this paper were carried out at the London School of Hygiene and Tropical Medicine (LS)> and the remainder at the Wellcome Laboratories for Experimental Parasitology, Glasgow. Rats. Female, random-bred Wistars were used throughout. At LS the rats were supplied commercially (A. J. Tuck & Co.) and used at 130-180 g. In Glasgow, rats were bred at the laboratory and used at 130-150 g. Strongyloides ratti. The strain of S. ratti used at LS was obtained from the Wellcome Laboratories, Beckenham, Kent. A similar, homogonic strain was used at GIasgow and was obtained from Dr. P. A. G. Wilson, Department of Zoology, University of Edinburgh. Third-stage ( LR), infective, filariform larvae were obtained by culturing infected feces, using either the test tube/filter paper method (Moqbel and Denham 1977) or the charcoal method (Zamirdin and Wilson 1974). Rats were infected by injecting larvae subcutaneously. Trichinella spiralis. The London School strain of T. spiralis was used throughout. Rats were infected with T. spiralis using the method described by Wakelin and Lloyd ( 1976). Recouwy of worms. Adult worms of both species were recovered by incubating the longitudinally slit intestine in either phosphate buffered saline (PBS) or Hanks’ balanced salt solution (BSS) at
Trichinella
spiralis AND Strongyloides
Strongyloides
ratti Infection
TABLE Effect Experiment
of an Initial Group
Initial
S. ratti infection
Day given
Number of larvae
I
on a Subsequent Number of T. spiralis given Day 0
1
1 2
-30 -
1000 Xone
1000 1000
2
1 2
-35 -
3000 None
2000 2000
67
ratti: INTERACTION
Challenge
with
T. spiralis Mean number
I’richir~ella
spiralis
recovered
in Rats
-
Day 7
Length (mm)
Fecundity L/ 0 /hr
22 83
1.2 f 0.8 1.78 f 0.4
ND” XD
442 zk 175 1271 f 146
1.16 f 0.4 2.12 h 0.3
1.2 3.2
50 * 614 f
a Not done.
37 C for about 3 hr (see Wakelin and Wilson 1977). Thirty worms per group were randomly selected and measured with the aid of a camera Lucida and a calibrated roller-head map measurer. Estimation of worm fecundity. Parasitic S. ratti worms are considered to be parthenogenetic females; their fecundity was assessed from the mean number of eggs in utero, based upon a sample of 30 randomly-selected worms, and from the number of larvae per gram of feces. Fecundity of T. spiralis was estimated by the in vitro culture method of Wakelin and Wilson (1977) and is expressed as the number of newborn larvae released per female per hour (L/?/hr). Anth-elmintic. Thiabendazole (TBZ) was used to remove S. ratti from infected rats. A dose of 100 mg/kg given on 2 consecutive days had an efficacy of 99 to 10070. Presentation of data. In the experiments described below, the day of T. spiralis challenge was considered as Day 0. This was in order to accommodate the staggered dates of the initial S. ratti infection. Worm recoveries are expressed as mean and standard deviation. RESULTS
Effect of an Initial Infection with on a Subsequent Challenge with T. Two experiments were carried which rats were challenged with T.
S. ratti spiralis out in spiralis
almost immediately after a full initial infection with S. ratti. The protocols and results of both experiments are given in Table I. In the first experiment (LS) five rats from Group 1 and four from Group 2 were killed on Day 7 after challenge with T. spiralis. Rats previously exposed to S. ratti harbored fewer adults on Day 7 and these worms were significantly shorter than control worms. In addition, in rats killed on Day 28 and digested individually it was found that there were fewer muscle larvae in the group exposed to a prior S. ratti infection (2360 * 1959 vs 27,259 + 20,000 larvae per rat). In the second experiment, five rats from each group were killed 7 days after T. spiralis challenge. Here again, rats exposed to the prior S. ratti infection harbored a reduced number of adult T. spiralis, which were shorter and less fecund than the control worms. In a further series of four experiments, challenge with T. spiralis was given a considerable period after rats had expelled their initial S. ratti burden to determine whether or not the premature expulsion of T. spiralis, observed earlier, was brought about by a specific cross-reaction between the two species. In the first experiment (LS) rats were challenged with T. spiralis 43 days after a full primary S. ratti infection; in the second experiment (LS) T. spiralis challenge was given 50 days after S. ratti; in the
68
MOQBEL
TABLE
AND
II
The Course of Delayed Challenge with I’richinella spiralis following an Initial Slrong~loides rattz InfectionS Experimerit
NUmbfX of rats
Initial Day given
8. ratti
infection
Number larrae
of
Mean 70 recovery of T. spiralis on Day 7
1
5 4
-43
2000 None
3.9 34.0
2
5 4
- 50 -
1000 None
13.6 435
3
5 5 6
-42 -62 -
1000 1000 I\Jone
5.3 14.6 66.1
4
5 5
- 55
2000 None
3.0 33.7 -
a All
groups
infected
with
1000
T. spiralis
on Day
0.
third (LS) 42 or 62 days after; and in the fourth rats were challenged 55 days after S. r&i. Each experiment contained a relevant challenge control group. The experimental designs and the results are summarized in Table II. Since different levels of T. spiralis challenge were used, the numbers of worms recovered on Day 7 are expressed as a percentage of the inoculum given. The results of these experiments confirmed that T. spiralis infections were not successful in rats previously infected with S. ratti when given 30 to 42 days after the expulsion of the initial S. rutti infection. These experiments suggest that a specific cross-immunity was probably operative, since the interval between the expulsion of S. ratti burden and T. spiralis challenge was long enough to eliminate the possibility of there being a nonspecific effect from inflammation in the small intestine. Effect of S. ratti on the Estaldishmerzt and/or Survival of T. spiralis Challenge The reduction in numbers of T. spiralis present 7 days after challenge in rats previously infected with S. ratti could result from an effect upon either establishment or the survival of the T. spiralis challenge. Twenty rats were divided into
WAKEXJN
two equal groups; rats in Group 1 were each given 1000 S. ratti and those in Group 2 were left uninfected. Both groups were treated with TBZ on Days 26 and 27 p.i. to remove the few sterile residual S. r&i and were challenged 9 days later with 1000 T. spiralis each, There was no significant difference between the number of T. spiralis recovered from Groups 1 and 2 3 days after challenge (mean: 380 _t 99 vs 459 * 116, respectively). However, on Day 8 after challenge a mean of only 1 k 1.1 worms was recovered from the challenged Group 1 while rats in Group 2 harbored a mean of 262 * 165 worms. It thus appears that the effect exerted by prior infection with S. rutti was operative against the survival of the T. spiralis challenge and did not affect its initial establishment. Reciprocity of Interaction and T. spiralis
between S. ratti
Three experiments were carried out to discover whether S. rntti can be established in rats which have recently expelled the intestinal stages of a primary T. spiralis infection. The course of S. ratti challenge was also examined in adult rats harboring fully developed T. spiralis muscle larvae. The protocol of the experiments and the results obtained are presented in Table III. The results of the first experiment showed that the response elicited by the intestinal phase of a previous T. spiralis infection markedly affected the development of the S. ratti challenge given 15 or 21 days later, as evidenced by the severe reduction in number of adult S. ratti recovered 13 days after challenge. In the second experiment, where the S. ratti challenge was delayed until Day 48 after T. spiralis, when the rats harbored a mature muscle larval burden, recovery of S. ratti on Day 15 again showed a marked reduction relative to the S. rntti controls, but
Trichinella
spiralis AND Strongyloides TABLE
Recovery
Experiment
ratti: INTERACTION
III
of Strongyloides ratti from Infections Given at Different an Initial Trichinella spiralis Infection
Number of rats
Timing Day 0
of infections
Times after
Mean number of S. ratti at autopsy after challenge
Day 15 Day 21 Day 48 Day 13
1
T. s.‘” -
5 4 6 5
2
S. I..~ *f r. -
T. s.
-
10
T. s.
5
T. s. s. r. -
1:
69
Day 32
-S. r.
-
78f 326 f
31 71
-
-
-
i.r. c
-
84 f 321 f
93 139
-
-
*9 r.
-
Day 15
T. s. s. r. Ls r.
-
-
182 f 103 0 3f3 446 f 119
31 f24 SD” ND
0
u T. spiralis, 1800 per rat groups 1 and 3; 1000 to groups 5 and 6. b S. r&i, 1000 per rat. c Not done.
on Day 32, when the latter had lost all their worms, the double infection group still retained a small number of S. ratti. The homologous challenge groups showed that the rats had developed immunity against T. spiralis and S. ratti in the normal manner. The effect of muscle larvae on a subsequent S. ratti infection was re-examined using an experimental design similar to the second experiment, except that the
challenge was given 37 days with T. spiralis and rats were killed on Days 15, 30, and 36 after S. ratti infection. The results also suggested a delayed expulsion of S. ratti from rats harboring mature muscle larval stages of T. spiralis, and this was reflected also in the size and fecundity of the worms recovered. At Day 36 after S. ratti challenge, worms in the double infection group were 1.71 * 1.1 mm long and had approximately four eggs per
TABLE Recovery
of Adult
Groups
1. T. spiralis
+ S. ratti
2. T. spiralis
alone
IV
Worms from the Small Intestine of Rats Concurrently with Trichinella spiralis and Strongyloides ratti Day of autopsy”
3. S. rutti alone
a At autopsy, six rats were killed * Three rats killed. c Four rats killed.
14 18 14b lSc 14 18
Trichinella ___ Mean number of adult worms 128 f 118 0 73 z!z 43 0
spiralis Fecundity L/ 0 /hr
1.2
Infected
Strongyloides Mean number of adult worms
ratti
Mean in utero egg/worm
0.2 -
146 z~z 78 226 + 109 -
5.3 f 3.7 4.2 f 2.9 -
-
437 f 565 f
9.6 f 5.2 7.7 It 5.5
-
per group except where noted.
91 123
70
MOQBEL
AND
female whereas worms in the S. rutti controls were only 0.91 * 0.9 mm long and had no eggs in their uteri. The Course of Concurrent T. spiralis and S. ratti Infections in the Rats The previous experiments examined direct immunity between these two species, with one nematode preceding the other and influencing the establishment of the heterologous challenge. In the following experiment we investigated the course of infection with both parasites and the effect each one exerts on the other when present concurrently in the host. Twenty-nine rats were arranged into three groups. Group 1 (12 rats) was infected with both T. spiralis (1800 larvae) and S. ratti (1000 larvae), Group 2 (7 rats) with T. spiraZis (1800) only, and Group 3 (12 rats) with S. ratti (1000) only, all on the same day. The numbers of adult S. ratti and T. spiralis and worm fecundities were determined on Days 14 and 18 p.i. and these results are summarized in Table IV. Feces of rats from Groups 1 and 3 (harboring S. ratti) were also cultured on Days 11 and 15 p.i. to determine changes in the larval production during the course of infection. In concurrently infected rats (Group 1 ), the expulsion of T. spiralis was slightly delayed compared with rats infected with T. spiralis alone (Group 2); on Day 14 p.i. the percentage recoveries were 12.8 and 7.170, respectively. This delay was also reflected in worm fecundity, which was markedly higher on Day 14 in Group 1 than in Group 2. No worms were recovered from either group on Day 18 p.i. The establishment of S. ratti, on the other hand, was affected in concurrently infected rats. Using Student’s t test it was evident that there was a significantly (P < 0.01) lower percentage recovery in these rats than in the controls on both days
WAKELIN
1114.5vs 43.6% on Day 14 p.i., and 22.6 vs 56.6% on Day 18 p.i.1. This severe reduction in worm burden in Group 1 was correlated with a marked reduction in the number of eggs per worm on both Days 14 and 18 and with the number of larvae/ gram of feces during the course of infection. Rats in Group 1 yielded 371 * 161 and 212 k 73 larvae/gram, while the controls (Group 3) produced 1588 * 188 and 3271 I+ 231 larvae/gram on Days 11 and 15, respectively. In a repeat experiment similar results were obtained, although on this occasion a small number of T. spiralis was present on Day 18 pi. in the concurrently infected rats, when the corresponding controls were worm free. The S. ratti picture, in rats given both parasites or S. ratti alone, was very similar to those obtained in the previous experiment. DISCUSSION The experiments described here suggest that the cross-reaction between S. ratti and T. spiralis has a direct immunological basis, as the survival of each was adversely affected when infections were given long after the host would have undergone spontaneous cure of the immunizing species. The fact that the interaction was reciprocal strengthens the possibility that it was immunologically mediated as does our observation that corticosteroids eliminated the manifestations of acquired immunity to both homologous and heterologous challenge ( Moqbel and Wakelin, unpublished). In concurrent infections the response to T. spiralis proved detrimental to S. ratti but no reciprocal effect was evident, presumably because the short survival of T. spiralis in the rat precluded any effect from the slower immune and inflammatory responses evoked by S. ratti. Cross-immunity involving S. ratti has also been reported by other workers. A reciprocal cross-immunity with Nippo-
Trichinella
Spirali.
AND
Strongyloides
strongylus bra&en& was described by Kazacos and Thorson (1975) and it is interesting that, as with T. spiralis (Table III), the immunity was less effective against S. ratii than against the other species involved. Both S. ratti and N. brasilieti infect the rat by skin penetration and reach the intestine via the lungs; it is therefore possible that the cross-immunity may have operated parenterally through nonspecific trapping of larvae in the lungs. However, immune precipitates were observed when larvae were incubated in heterologous immune sera and Kazacos and Thorson (1975) speculated that antibodies against common penetration enzymes may be involved. A further instance of cross-immunity was described by Kazacos (1976) using S. rutti as the challenging species in rats immunized against T. spir&s. No cross-reactions were observed when larvae were incubated in immune sera and Kazacos postulated that crossimmunity might be mediated through the intestinal inflammation evoked by T. viralis, although this seems unlikely from the experimental design. The nature of the responses involved in cross-immunity between S. r&i and T. spiralis remains an intriguing problem. Rats immunized against T. spiralis are capable of a rapid response to homologous challenge and worm expulsion may occur very rapidly-within 24 hr. In contrast, in rats immunized against S. ratti there was no immediate effect upon the establishment of T. spiralis; cross-immunity was operative against worm survival. Russell and Castro (1977) have suggested that challenge immunity against T. spiralis may involve mechanisms distinct from those responsible for spontaneous cure during a primary infection. It seems unlikely that this is true of immunity elicited by prior S. ratti infection, where expulsion appears as an accelerated version of the primary response. Two mechanisms can be proposed to explain this. First, lymphocytes
mtti:
INTERACTION
71
primed by exposure to S. ratti respond to T. spiralis and initiate the series of events, both immunological and inflammatory, which results in the expulsion of T. spiralis. Alternatively, anti-S. ratti antibodies may cross-react with T. spiralis, reducing their ability to survive the intestinal response which the challenge itself evokes. Analysis of the reciprocal situation is complicated by the fact that S. ratti has a parenteral migration before reaching the intestine. In secondary infections-with this species immunity operates during lung migration to reduce the numbers of worms reaching the intestine (Moqbel 1977). Cross-immunity from prior T. spirulis infection may therefore also be operative parenterally, possibly as a result of the enhanced cellular immunity which follows exposure to T. spiralis (Cypess et al. 1974; Meerovitch and Bomford 1977); a similar proposal has been made by Kazacos ( 1976). There is almost certainly an element of specificity in the cross-immunity between S. ratti and T. spiralis, since, whereas rats immunized against the latter show marked resistance to S. ratii (this paper and Kazacos 1976), rats similarly immunized show only slight resistance to N. bmsiliensis, which also migrates through the lungs. The possibility that the cross-immunity is operative also at the intestinal level still exists and requires further investigation. Although the emphasis in this study was upon cross-resistance it is well known that immunological interactions between nematodes may involve immunodepression by one species enhancing the survival of another (Jenkins and Behnke 1977). It is interesting therefore to note that there was some indication that complete loss of S. ratti from rats carrying muscle larvae of T. spiralis was delayed relative to that in control rats, even though earlier development and survival was impaired. T. spiralis infections are known to exert a degree of immunodepression (Faubert 1976; Ljungstrom and Huldt 1977) and
MOQBEL AND WAKELIN
72
it is possible that the interaction between T. spiralis and S. ratti involves the interplay of cross-immunity and cross-suppression. ACKNOWLEDGMENTS We would like to acknowledge the cooperation of Miss N. A. Moloney (L.S.H.T.M.) during the early part of this work. Our thanks to Miss M. M. Wilson for her technical assistance and to Mr. Jack Keys for supervising the maintenance of our animals. This work was supported by the Wellcome Trust (R. M.) and the Medical Research Council (D. W., Project Grant G977/ 65/T).
REFERENCES BEHNKE, J. M., BLAND, P. W., AXD WAKELIX, D. 1977. Effect of the expulsion phase of Trichinella spiralis on Hymenolepis diminuta infection in mice. Parasitology 75, 79-88. BRUCE, R. G., AsD WAKELIN, D. 1977. Immunological interactions between Trichinellu s~Gdis and Trichuris murk in the intestine of the mouse. Parasitology 74, 163-173. CYPESS, R. H., LUBINIECKI, A. S., AND SWIDU'A, D. M. 1974. Decreased susceptibility to Listeria monocytogenes in mice after infection with Trichinella spiralis. Infection and Immunity 9, 477-479. FAUBERT, C. E. 1976. Depression of the plaqueforming cells to sheep red blood cells by the new-born larvae of Trichinellu spiralis. Immunology 30, 485-489. JENKINS, S. N., AXD BEHNKE, J. M. 1977. Impairment of primary expulsion of Trichuris concurrently infected with murk in mice Nematospiroides dub&. Parasitology 75, 71-78. KAZACOS, K. R. 1975. Increased resistance in the rat to Nippostrongylus brusiliensis following immunization against Trichinella spiralis. Veterinary Parasitology 1, 165-174. KAZACOS, K. R. 1976. Increased resistance in the rat to Strongyloides ratti following immunization against Trichin&a spiralis. Journal of Parasitology 62, 493-494. KAZACOS, K. R., AND THORSON, R. E. 1975. Crossresistance between Nippostrongylus brusiliensb and Strongyl0ide.s rutti in rats. ~mrnul of Parasitology 61, 525-529. LARSH, J. E., JR., ASD RACE, G. J. 1975. Allergic inflammation as a hypothesis for the expulsion
of worms from tissues. A review. Parasitology 37, 251-26’6.
LJIJYXSTRCI~, I.,
Experimental
AND HULDT, G. 1977. Effect of experimental Trichinosis on unrelated humoral and cell mediated immunity. Acta Pathologica Microbiologica Scandinavica, Section C, 85, 131-141. LOVE, R. J., OGILVIE, B. M., ASD MCLAREN, D. J. 1976. The immune mechanism which expels the intestinal stage of Trichinellu spiralis from rats. Immunology 30, 7-15. MCCOY, 0. R. 1931. Immunity of rats to reinfection with Trichinelln spiralis. American Journal of Hygiene 14, 484-494. MEEROVITCH, E., AND BOhrFORD, R. 1977. Macrophage potent&ion by TriclzinelLu spin&. Annals of Tropicul Medicine and Parasitology 71, 245-249. MOQUEL, R. 1977. “Studies on the Host-Parasite Relationship of Strongyloides r&i in Rats.” Ph.D. thesis, University of London. MOQHEL, R., AND DENITAXI, D. A. 1977. Strongyloides rutti: 1. Parasitological observations on primary and secondary infections in the small intestine of rats. Journal of Helminthology 51, 301-308. MOQBEL, R., AND ELLIS, D. S. 1978. Effect of the immune response of rats on adult StrongyZoides ratti. Parusitology (Abstract) 77, xIvii. RUSSELL, D., AXD CASTRO, C. A. 1977. A biphasic response to explain immune rejection of Trichinellu spiralis from the rat intestine. “Abstract ( 161), 52nd Annual Meeting of the American Society of Parasitologists, Nevada, 1977.” STEWART, D. F. 1953. Studies on resistance of sheep to infestation with Haemonchus contortus and Trichostrongylus spp. and on the immunological reactions of sheep exposed to infestation. V. The nature of the “self-cure” phenomenon. Australian Journal of Agricultural Research 4, 100-l 17. WAKELIN, D., AXD LLOYD, M. 1976. Immunity to primary and challenge infections of Trichinella spiralis in mice: A re-examination of conventional parameters. Parasitology 72, 173-182. WAKELIN, D., AND WILSOS, M. M. 1977. Transfer of immunity to Trichinella spiralis in the mouse with mesenteric lymph node celIs: Time of appearance of effective cells in donors and expression of immnnity in recipients. Pnrasitology 74, 215-224. ZAMIRDIN, M., AND WILSON, P. A. C. 1974. Strongyloides r&i: Relative importance of maternal sources of infection. ParaGtoZogy 69, 445-453.
PARASITOLOGY
47, 65-72
(1979)
Trichinella spiralis and Sfrongyloides ratti: Immune Interaction in Adult Rats R. MOQBEL AND D. WAKELIN Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Bearsden Road, Bearsden, Glasgow, G61 1 QH, Scotland, United Kingdom (Accepted
for publication
I7 October
1978)
MOQBEL, R., AND WAKELIN, D. 1979. Trichinella spiralis and Strongyloides ratti: Immune interaction in adult rats. Experimenta Parasitology 47, 65-72. The consequences of prior and concurrent infection with two species of nematodes were studied in rats. Primary infection with Strongyloides ratti adversely affected the development of a secondary Trichinella spiralis infection. Both immediate and delayed challenge with T. spiralis, following the expulsion of the previous S. ratti infection, reduced the percentage of worm recovery of the former as well as their fecundities and lengths. It is suggested that nonspecific inflammation produced by one species, during the peak period of worm expulsion, was not responsible for the accelerated rate of expulsion of the other; instead a direct, specific cross-immunity was probably operative affecting the survival of the challenge species. The response elicited by previous experience of the intestinal phase was reciprocal, but there was evidence of an enhancing effect by the muscle larval stages of T. spiralis on S. ratti. Rats concurrently infected with both species expelled S. ratti more rapidly than T. spiralis. Possible mechanisms underlying the interaction between the two species are suggested and discussed. INDEX DESCRIPTORS: Trichinella spiralis; Strongyloides ratti; Cross-immunity; Concurrent infections; Immune expulsion; Intestinal inflammation; Immunosuppression; Rat; Nematode.
ever, in the majority of cases, the crossimmunity arises indirectly from the immune response, most commonly as a result of the intestinal “allergic” inflammation (reviewed by Larsh and Race 1975) evoked by one species acting nonspecifically against the other species present (Stewart 1953; Bruce and Wakelin 1977; Behnke et al. 1977). Direct and indirect cross-immunity can be differentiated when infection with the challenging species is given a long time after the initial infection, i.e., when the intestinal environment has returned to normal and the manifestations of the inflammatory response have subsided.
The phenomenon of “cross-immunity” between species of intestinal helminths has been widely studied because of its potential significance in the context of naturally occurring infections, both in man and domestic animals. In a number of species combinations, prior or concurrent infection with one species will adversely affect the establishment or survival of a second (reviewed by Kazacos 1975). It has often been assumed that this effect has a direct immunological basis, i.e., the species concerned share common antigens and the immune response evoked by one species operates directly against the other. How65
0014-4894/79/010065-08$02.00/O All
Copyright 0 1979 rights of reproduction
by
Academic Press, Inc. in any form reserved.
MOQBEL
AND WAKELIN
between T. spirulis nld S. rutti h rats, using an initial infection with one species as the source of sensitization against the other; (ii) the reciprocity of the response; (iii) the effect of the acute (intestinal phase) and chronic (muscle larval phase) stages of T. spiralis 011 the course of S. ratti infection; (iv) the course of concurrent infections with both species in rats. MATEHIALS
I
1”
DAYS AFTER
2”
INFECTION
FIG. 1. Schematic diagram of the course of primary and secondary infections with Trichinellu spidis (open bars) and Strongyloides ratti (closed bars) in adult rats (t) Day of secondary infection.
The two nematodes studied here evoke strong immunity in the rat host. Rats initially infected with Strongyloicles ratti or TrichineEZa spiralis manifest the phenomenon of spontaneous cure (Fig. 1) and subsequently exhibit strong resistance to reinfection (Moqbel and Denham 1977; Love et al. 1976; McCoy 1931). During primary and secondary infections with either species, resistance is reflected in changes in worm fecundity and morphology as well as in the position occupied in the small intestine (Moqbel and Ellis 1978; Love et al. 1976). Immunity to T. spiralis is associated with marked intestinal inflammation. S. ratti, on the other hand, induces a number of histopathological changes in the small intestine (Moqbel 1977), but the degree of inflammation is much less pronounced than in rats infected with T. spiralis. The experiments described in this report were designed to investigate the folIowing points: (i) th e nature of cross-reaction
AND METHODS
Some of the experiments described in this paper were carried out at the London School of Hygiene and Tropical Medicine (LS)> and the remainder at the Wellcome Laboratories for Experimental Parasitology, Glasgow. Rats. Female, random-bred Wistars were used throughout. At LS the rats were supplied commercially (A. J. Tuck & Co.) and used at 130-180 g. In Glasgow, rats were bred at the laboratory and used at 130-150 g. Strongyloides ratti. The strain of S. ratti used at LS was obtained from the Wellcome Laboratories, Beckenham, Kent. A similar, homogonic strain was used at GIasgow and was obtained from Dr. P. A. G. Wilson, Department of Zoology, University of Edinburgh. Third-stage ( LR), infective, filariform larvae were obtained by culturing infected feces, using either the test tube/filter paper method (Moqbel and Denham 1977) or the charcoal method (Zamirdin and Wilson 1974). Rats were infected by injecting larvae subcutaneously. Trichinella spiralis. The London School strain of T. spiralis was used throughout. Rats were infected with T. spiralis using the method described by Wakelin and Lloyd ( 1976). Recouwy of worms. Adult worms of both species were recovered by incubating the longitudinally slit intestine in either phosphate buffered saline (PBS) or Hanks’ balanced salt solution (BSS) at
Trichinella
spiralis AND Strongyloides
Strongyloides
ratti Infection
TABLE Effect Experiment
of an Initial Group
Initial
S. ratti infection
Day given
Number of larvae
I
on a Subsequent Number of T. spiralis given Day 0
1
1 2
-30 -
1000 Xone
1000 1000
2
1 2
-35 -
3000 None
2000 2000
67
ratti: INTERACTION
Challenge
with
T. spiralis Mean number
I’richir~ella
spiralis
recovered
in Rats
-
Day 7
Length (mm)
Fecundity L/ 0 /hr
22 83
1.2 f 0.8 1.78 f 0.4
ND” XD
442 zk 175 1271 f 146
1.16 f 0.4 2.12 h 0.3
1.2 3.2
50 * 614 f
a Not done.
37 C for about 3 hr (see Wakelin and Wilson 1977). Thirty worms per group were randomly selected and measured with the aid of a camera Lucida and a calibrated roller-head map measurer. Estimation of worm fecundity. Parasitic S. ratti worms are considered to be parthenogenetic females; their fecundity was assessed from the mean number of eggs in utero, based upon a sample of 30 randomly-selected worms, and from the number of larvae per gram of feces. Fecundity of T. spiralis was estimated by the in vitro culture method of Wakelin and Wilson (1977) and is expressed as the number of newborn larvae released per female per hour (L/?/hr). Anth-elmintic. Thiabendazole (TBZ) was used to remove S. ratti from infected rats. A dose of 100 mg/kg given on 2 consecutive days had an efficacy of 99 to 10070. Presentation of data. In the experiments described below, the day of T. spiralis challenge was considered as Day 0. This was in order to accommodate the staggered dates of the initial S. ratti infection. Worm recoveries are expressed as mean and standard deviation. RESULTS
Effect of an Initial Infection with on a Subsequent Challenge with T. Two experiments were carried which rats were challenged with T.
S. ratti spiralis out in spiralis
almost immediately after a full initial infection with S. ratti. The protocols and results of both experiments are given in Table I. In the first experiment (LS) five rats from Group 1 and four from Group 2 were killed on Day 7 after challenge with T. spiralis. Rats previously exposed to S. ratti harbored fewer adults on Day 7 and these worms were significantly shorter than control worms. In addition, in rats killed on Day 28 and digested individually it was found that there were fewer muscle larvae in the group exposed to a prior S. ratti infection (2360 * 1959 vs 27,259 + 20,000 larvae per rat). In the second experiment, five rats from each group were killed 7 days after T. spiralis challenge. Here again, rats exposed to the prior S. ratti infection harbored a reduced number of adult T. spiralis, which were shorter and less fecund than the control worms. In a further series of four experiments, challenge with T. spiralis was given a considerable period after rats had expelled their initial S. ratti burden to determine whether or not the premature expulsion of T. spiralis, observed earlier, was brought about by a specific cross-reaction between the two species. In the first experiment (LS) rats were challenged with T. spiralis 43 days after a full primary S. ratti infection; in the second experiment (LS) T. spiralis challenge was given 50 days after S. ratti; in the
68
MOQBEL
TABLE
AND
II
The Course of Delayed Challenge with I’richinella spiralis following an Initial Slrong~loides rattz InfectionS Experimerit
NUmbfX of rats
Initial Day given
8. ratti
infection
Number larrae
of
Mean 70 recovery of T. spiralis on Day 7
1
5 4
-43
2000 None
3.9 34.0
2
5 4
- 50 -
1000 None
13.6 435
3
5 5 6
-42 -62 -
1000 1000 I\Jone
5.3 14.6 66.1
4
5 5
- 55
2000 None
3.0 33.7 -
a All
groups
infected
with
1000
T. spiralis
on Day
0.
third (LS) 42 or 62 days after; and in the fourth rats were challenged 55 days after S. r&i. Each experiment contained a relevant challenge control group. The experimental designs and the results are summarized in Table II. Since different levels of T. spiralis challenge were used, the numbers of worms recovered on Day 7 are expressed as a percentage of the inoculum given. The results of these experiments confirmed that T. spiralis infections were not successful in rats previously infected with S. ratti when given 30 to 42 days after the expulsion of the initial S. rutti infection. These experiments suggest that a specific cross-immunity was probably operative, since the interval between the expulsion of S. ratti burden and T. spiralis challenge was long enough to eliminate the possibility of there being a nonspecific effect from inflammation in the small intestine. Effect of S. ratti on the Estaldishmerzt and/or Survival of T. spiralis Challenge The reduction in numbers of T. spiralis present 7 days after challenge in rats previously infected with S. ratti could result from an effect upon either establishment or the survival of the T. spiralis challenge. Twenty rats were divided into
WAKEXJN
two equal groups; rats in Group 1 were each given 1000 S. ratti and those in Group 2 were left uninfected. Both groups were treated with TBZ on Days 26 and 27 p.i. to remove the few sterile residual S. r&i and were challenged 9 days later with 1000 T. spiralis each, There was no significant difference between the number of T. spiralis recovered from Groups 1 and 2 3 days after challenge (mean: 380 _t 99 vs 459 * 116, respectively). However, on Day 8 after challenge a mean of only 1 k 1.1 worms was recovered from the challenged Group 1 while rats in Group 2 harbored a mean of 262 * 165 worms. It thus appears that the effect exerted by prior infection with S. rutti was operative against the survival of the T. spiralis challenge and did not affect its initial establishment. Reciprocity of Interaction and T. spiralis
between S. ratti
Three experiments were carried out to discover whether S. rntti can be established in rats which have recently expelled the intestinal stages of a primary T. spiralis infection. The course of S. ratti challenge was also examined in adult rats harboring fully developed T. spiralis muscle larvae. The protocol of the experiments and the results obtained are presented in Table III. The results of the first experiment showed that the response elicited by the intestinal phase of a previous T. spiralis infection markedly affected the development of the S. ratti challenge given 15 or 21 days later, as evidenced by the severe reduction in number of adult S. ratti recovered 13 days after challenge. In the second experiment, where the S. ratti challenge was delayed until Day 48 after T. spiralis, when the rats harbored a mature muscle larval burden, recovery of S. ratti on Day 15 again showed a marked reduction relative to the S. rntti controls, but
Trichinella
spiralis AND Strongyloides TABLE
Recovery
Experiment
ratti: INTERACTION
III
of Strongyloides ratti from Infections Given at Different an Initial Trichinella spiralis Infection
Number of rats
Timing Day 0
of infections
Times after
Mean number of S. ratti at autopsy after challenge
Day 15 Day 21 Day 48 Day 13
1
T. s.‘” -
5 4 6 5
2
S. I..~ *f r. -
T. s.
-
10
T. s.
5
T. s. s. r. -
1:
69
Day 32
-S. r.
-
78f 326 f
31 71
-
-
-
i.r. c
-
84 f 321 f
93 139
-
-
*9 r.
-
Day 15
T. s. s. r. Ls r.
-
-
182 f 103 0 3f3 446 f 119
31 f24 SD” ND
0
u T. spiralis, 1800 per rat groups 1 and 3; 1000 to groups 5 and 6. b S. r&i, 1000 per rat. c Not done.
on Day 32, when the latter had lost all their worms, the double infection group still retained a small number of S. ratti. The homologous challenge groups showed that the rats had developed immunity against T. spiralis and S. ratti in the normal manner. The effect of muscle larvae on a subsequent S. ratti infection was re-examined using an experimental design similar to the second experiment, except that the
challenge was given 37 days with T. spiralis and rats were killed on Days 15, 30, and 36 after S. ratti infection. The results also suggested a delayed expulsion of S. ratti from rats harboring mature muscle larval stages of T. spiralis, and this was reflected also in the size and fecundity of the worms recovered. At Day 36 after S. ratti challenge, worms in the double infection group were 1.71 * 1.1 mm long and had approximately four eggs per
TABLE Recovery
of Adult
Groups
1. T. spiralis
+ S. ratti
2. T. spiralis
alone
IV
Worms from the Small Intestine of Rats Concurrently with Trichinella spiralis and Strongyloides ratti Day of autopsy”
3. S. rutti alone
a At autopsy, six rats were killed * Three rats killed. c Four rats killed.
14 18 14b lSc 14 18
Trichinella ___ Mean number of adult worms 128 f 118 0 73 z!z 43 0
spiralis Fecundity L/ 0 /hr
1.2
Infected
Strongyloides Mean number of adult worms
ratti
Mean in utero egg/worm
0.2 -
146 z~z 78 226 + 109 -
5.3 f 3.7 4.2 f 2.9 -
-
437 f 565 f
9.6 f 5.2 7.7 It 5.5
-
per group except where noted.
91 123
70
MOQBEL
AND
female whereas worms in the S. rutti controls were only 0.91 * 0.9 mm long and had no eggs in their uteri. The Course of Concurrent T. spiralis and S. ratti Infections in the Rats The previous experiments examined direct immunity between these two species, with one nematode preceding the other and influencing the establishment of the heterologous challenge. In the following experiment we investigated the course of infection with both parasites and the effect each one exerts on the other when present concurrently in the host. Twenty-nine rats were arranged into three groups. Group 1 (12 rats) was infected with both T. spiralis (1800 larvae) and S. ratti (1000 larvae), Group 2 (7 rats) with T. spiraZis (1800) only, and Group 3 (12 rats) with S. ratti (1000) only, all on the same day. The numbers of adult S. ratti and T. spiralis and worm fecundities were determined on Days 14 and 18 p.i. and these results are summarized in Table IV. Feces of rats from Groups 1 and 3 (harboring S. ratti) were also cultured on Days 11 and 15 p.i. to determine changes in the larval production during the course of infection. In concurrently infected rats (Group 1 ), the expulsion of T. spiralis was slightly delayed compared with rats infected with T. spiralis alone (Group 2); on Day 14 p.i. the percentage recoveries were 12.8 and 7.170, respectively. This delay was also reflected in worm fecundity, which was markedly higher on Day 14 in Group 1 than in Group 2. No worms were recovered from either group on Day 18 p.i. The establishment of S. ratti, on the other hand, was affected in concurrently infected rats. Using Student’s t test it was evident that there was a significantly (P < 0.01) lower percentage recovery in these rats than in the controls on both days
WAKELIN
1114.5vs 43.6% on Day 14 p.i., and 22.6 vs 56.6% on Day 18 p.i.1. This severe reduction in worm burden in Group 1 was correlated with a marked reduction in the number of eggs per worm on both Days 14 and 18 and with the number of larvae/ gram of feces during the course of infection. Rats in Group 1 yielded 371 * 161 and 212 k 73 larvae/gram, while the controls (Group 3) produced 1588 * 188 and 3271 I+ 231 larvae/gram on Days 11 and 15, respectively. In a repeat experiment similar results were obtained, although on this occasion a small number of T. spiralis was present on Day 18 pi. in the concurrently infected rats, when the corresponding controls were worm free. The S. ratti picture, in rats given both parasites or S. ratti alone, was very similar to those obtained in the previous experiment. DISCUSSION The experiments described here suggest that the cross-reaction between S. ratti and T. spiralis has a direct immunological basis, as the survival of each was adversely affected when infections were given long after the host would have undergone spontaneous cure of the immunizing species. The fact that the interaction was reciprocal strengthens the possibility that it was immunologically mediated as does our observation that corticosteroids eliminated the manifestations of acquired immunity to both homologous and heterologous challenge ( Moqbel and Wakelin, unpublished). In concurrent infections the response to T. spiralis proved detrimental to S. ratti but no reciprocal effect was evident, presumably because the short survival of T. spiralis in the rat precluded any effect from the slower immune and inflammatory responses evoked by S. ratti. Cross-immunity involving S. ratti has also been reported by other workers. A reciprocal cross-immunity with Nippo-
Trichinella
Spirali.
AND
Strongyloides
strongylus bra&en& was described by Kazacos and Thorson (1975) and it is interesting that, as with T. spiralis (Table III), the immunity was less effective against S. ratii than against the other species involved. Both S. ratti and N. brasilieti infect the rat by skin penetration and reach the intestine via the lungs; it is therefore possible that the cross-immunity may have operated parenterally through nonspecific trapping of larvae in the lungs. However, immune precipitates were observed when larvae were incubated in heterologous immune sera and Kazacos and Thorson (1975) speculated that antibodies against common penetration enzymes may be involved. A further instance of cross-immunity was described by Kazacos (1976) using S. rutti as the challenging species in rats immunized against T. spir&s. No cross-reactions were observed when larvae were incubated in immune sera and Kazacos postulated that crossimmunity might be mediated through the intestinal inflammation evoked by T. viralis, although this seems unlikely from the experimental design. The nature of the responses involved in cross-immunity between S. r&i and T. spiralis remains an intriguing problem. Rats immunized against T. spiralis are capable of a rapid response to homologous challenge and worm expulsion may occur very rapidly-within 24 hr. In contrast, in rats immunized against S. ratti there was no immediate effect upon the establishment of T. spiralis; cross-immunity was operative against worm survival. Russell and Castro (1977) have suggested that challenge immunity against T. spiralis may involve mechanisms distinct from those responsible for spontaneous cure during a primary infection. It seems unlikely that this is true of immunity elicited by prior S. ratti infection, where expulsion appears as an accelerated version of the primary response. Two mechanisms can be proposed to explain this. First, lymphocytes
mtti:
INTERACTION
71
primed by exposure to S. ratti respond to T. spiralis and initiate the series of events, both immunological and inflammatory, which results in the expulsion of T. spiralis. Alternatively, anti-S. ratti antibodies may cross-react with T. spiralis, reducing their ability to survive the intestinal response which the challenge itself evokes. Analysis of the reciprocal situation is complicated by the fact that S. ratti has a parenteral migration before reaching the intestine. In secondary infections-with this species immunity operates during lung migration to reduce the numbers of worms reaching the intestine (Moqbel 1977). Cross-immunity from prior T. spirulis infection may therefore also be operative parenterally, possibly as a result of the enhanced cellular immunity which follows exposure to T. spiralis (Cypess et al. 1974; Meerovitch and Bomford 1977); a similar proposal has been made by Kazacos ( 1976). There is almost certainly an element of specificity in the cross-immunity between S. ratti and T. spiralis, since, whereas rats immunized against the latter show marked resistance to S. ratii (this paper and Kazacos 1976), rats similarly immunized show only slight resistance to N. bmsiliensis, which also migrates through the lungs. The possibility that the cross-immunity is operative also at the intestinal level still exists and requires further investigation. Although the emphasis in this study was upon cross-resistance it is well known that immunological interactions between nematodes may involve immunodepression by one species enhancing the survival of another (Jenkins and Behnke 1977). It is interesting therefore to note that there was some indication that complete loss of S. ratti from rats carrying muscle larvae of T. spiralis was delayed relative to that in control rats, even though earlier development and survival was impaired. T. spiralis infections are known to exert a degree of immunodepression (Faubert 1976; Ljungstrom and Huldt 1977) and
MOQBEL AND WAKELIN
72
it is possible that the interaction between T. spiralis and S. ratti involves the interplay of cross-immunity and cross-suppression. ACKNOWLEDGMENTS We would like to acknowledge the cooperation of Miss N. A. Moloney (L.S.H.T.M.) during the early part of this work. Our thanks to Miss M. M. Wilson for her technical assistance and to Mr. Jack Keys for supervising the maintenance of our animals. This work was supported by the Wellcome Trust (R. M.) and the Medical Research Council (D. W., Project Grant G977/ 65/T).
REFERENCES BEHNKE, J. M., BLAND, P. W., AXD WAKELIX, D. 1977. Effect of the expulsion phase of Trichinella spiralis on Hymenolepis diminuta infection in mice. Parasitology 75, 79-88. BRUCE, R. G., AsD WAKELIN, D. 1977. Immunological interactions between Trichinellu s~Gdis and Trichuris murk in the intestine of the mouse. Parasitology 74, 163-173. CYPESS, R. H., LUBINIECKI, A. S., AND SWIDU'A, D. M. 1974. Decreased susceptibility to Listeria monocytogenes in mice after infection with Trichinella spiralis. Infection and Immunity 9, 477-479. FAUBERT, C. E. 1976. Depression of the plaqueforming cells to sheep red blood cells by the new-born larvae of Trichinellu spiralis. Immunology 30, 485-489. JENKINS, S. N., AXD BEHNKE, J. M. 1977. Impairment of primary expulsion of Trichuris concurrently infected with murk in mice Nematospiroides dub&. Parasitology 75, 71-78. KAZACOS, K. R. 1975. Increased resistance in the rat to Nippostrongylus brusiliensis following immunization against Trichinella spiralis. Veterinary Parasitology 1, 165-174. KAZACOS, K. R. 1976. Increased resistance in the rat to Strongyloides ratti following immunization against Trichin&a spiralis. Journal of Parasitology 62, 493-494. KAZACOS, K. R., AND THORSON, R. E. 1975. Crossresistance between Nippostrongylus brusiliensb and Strongyl0ide.s rutti in rats. ~mrnul of Parasitology 61, 525-529. LARSH, J. E., JR., ASD RACE, G. J. 1975. Allergic inflammation as a hypothesis for the expulsion
of worms from tissues. A review. Parasitology 37, 251-26’6.
LJIJYXSTRCI~, I.,
Experimental
AND HULDT, G. 1977. Effect of experimental Trichinosis on unrelated humoral and cell mediated immunity. Acta Pathologica Microbiologica Scandinavica, Section C, 85, 131-141. LOVE, R. J., OGILVIE, B. M., ASD MCLAREN, D. J. 1976. The immune mechanism which expels the intestinal stage of Trichinellu spiralis from rats. Immunology 30, 7-15. MCCOY, 0. R. 1931. Immunity of rats to reinfection with Trichinelln spiralis. American Journal of Hygiene 14, 484-494. MEEROVITCH, E., AND BOhrFORD, R. 1977. Macrophage potent&ion by TriclzinelLu spin&. Annals of Tropicul Medicine and Parasitology 71, 245-249. MOQUEL, R. 1977. “Studies on the Host-Parasite Relationship of Strongyloides r&i in Rats.” Ph.D. thesis, University of London. MOQHEL, R., AND DENITAXI, D. A. 1977. Strongyloides rutti: 1. Parasitological observations on primary and secondary infections in the small intestine of rats. Journal of Helminthology 51, 301-308. MOQBEL, R., AND ELLIS, D. S. 1978. Effect of the immune response of rats on adult StrongyZoides ratti. Parusitology (Abstract) 77, xIvii. RUSSELL, D., AXD CASTRO, C. A. 1977. A biphasic response to explain immune rejection of Trichinellu spiralis from the rat intestine. “Abstract ( 161), 52nd Annual Meeting of the American Society of Parasitologists, Nevada, 1977.” STEWART, D. F. 1953. Studies on resistance of sheep to infestation with Haemonchus contortus and Trichostrongylus spp. and on the immunological reactions of sheep exposed to infestation. V. The nature of the “self-cure” phenomenon. Australian Journal of Agricultural Research 4, 100-l 17. WAKELIN, D., AXD LLOYD, M. 1976. Immunity to primary and challenge infections of Trichinella spiralis in mice: A re-examination of conventional parameters. Parasitology 72, 173-182. WAKELIN, D., AND WILSOS, M. M. 1977. Transfer of immunity to Trichinella spiralis in the mouse with mesenteric lymph node celIs: Time of appearance of effective cells in donors and expression of immnnity in recipients. Pnrasitology 74, 215-224. ZAMIRDIN, M., AND WILSON, P. A. C. 1974. Strongyloides r&i: Relative importance of maternal sources of infection. ParaGtoZogy 69, 445-453.