STRONGYLOIDES RATTI INFECTIONS IN CONGENITALLY HYPOTHYMIC (NUDE) MICE

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1 Aust. J. Exp. Biul. Med. Sci., 0 (Pt.2) (1982) STRONGYLOIDES RATTI INFECTIONS IN CONGENITALLY HYPOTHYMIC (NUDE) MICE by H. J. S. DAWKINS, G. F. MITCHELL^' AND D. I. GROVE (From the Department of Medicine, University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 009, and *Laboratory of Immunoparasitology, The Walter and Eliza Hall Institute for Medical Research, Melbourne, Victoria 3050.) (Aceepted for publication January 15, 1982.) Summary. The course of infection with Strongyloides ratti was examined in congenitally hypothymic CBA/H, C57B1/ and BALB/c nude mice. The intensity of infection and the duration of faecal larval excretion were both increased in nude mice when compared with intact mice. Adull worms persisted in the small intestine of nude mice for at least weeks. Greater worm burdens were found in slich mice after subcutaneous injection as compared wiih percutaneous infection. Hypothymic mice did not acquire resistance to re-infection. It is concluded that both the spontaneous expulsion of worms in primary infection and resistance to challenge infection are T cell-dependent events. Autoinfection was not seen. INTRODUCTION Strongyloidiasis is one of the major human intestinal nematode infections. Unfortunately, Strongyloides stercoralis is not well adapted to small laboratory animals (Galliard, 197; Dawkins and Grove, 1982). Consequently, the closely related species, 5. ratti, has been used in experimental models of this infection. We have recently developed a murine model for strongyloidiasis (Dawkins et al, 1980) and shown that mice acquire a profound resistance to re-infection (Dawkins and Grove, 1981a). Resistance can be transferred by immune serum and cells (Dawkins and Grove, 1981b) and is partially abrogated by immunosuppression with corticosteroids (Grove and Dawkins, 1981). In this study we examine the kinetics of primary and secondary infections with S. ratti in hypothymic mice. Thus, the role of T cell-mediated events in the spontaneous expulsion of worms which occurs in primary infections and in the development of resistance to challenge infections is further delineated. MATERIALS AND METHODS Animals Young adult female CBA/H, C57B1/ and BALB/c congenitally hypothymic nu/nu (nude) mice and their heterozygous littermates (nu/4-) or normal ( + /-\-) animals were List of abbreviations used in this paper: N.S.. not significant, SD, Sprague-Dawley; S.D.. standard deviation, s.e, subcutaneously, p.c. percutaneously.

2 182 H. J. S. DAWKINS, G. F. MITCHELL AND D. I. GROVE supplied by the Walter and Eliza Hall Institute for Medical Research (Melbourne, Vic). Female Sprague-Dawley (SD) rats, g in weight, were supplied by the Animal Breeding Unit, University of Western Australia (Perth, W.A.). Strongyloides ratti An homogonlc Strain of S. ratti (originally obtained from Dr. P. A. G. Wilson, University of Edinburgh) was maintained by serial passage in SD rats. Methods for infecting mice both percutaneously (p.c.) and subcutaneousiy (s.c.) and for quantifying adult worms in the intestine, their fecundity and the numbers of larvae in the faeces have been described previously (Dawkins et al, 1980; Dawkins and Grove, 1981a). Statistics Student's t-test was used for all calculations. RESULTS Faecal larvae in primary infections Ten female CBA/H hypothymic (nu/nu) mice and 10 female intaet animals were infected s.c. with 400 filariform larvae of 5. ratti. The faeces from these animals were examined at various intervals after infection (Fig. 1). In the control mice, the kinetics of faecal larval excretion followed that usually seen in nonnal mice. Peak numbers of larvae were seen days after infection. This was followed by a rapid drop in numbers, with no larvae IS 20 X 3 40 TIME AFTER INFECTION (DAVSt Fig. 1. Faecal larval excretion in hypothymic CBA/H (nu/nu) mice ( ) and in intact control mice (O O) at various intervals after infection. There were 10 mice per group and the results are expressed as geometric mean ± S.E.M.

3 STRONGYLOIDIASIS IN NUDE MICE 183 being observed 12 days after infection. The faecal larval excretion of the nude miee was not signifieantly different from that of the intact animals when examined days after infection. When counted on days 9 and 12, however, the faecal larval numbers had increased two-fold (p < in each instance when compared with the faecal larvae in the same animals days after infection). Thereafter, there was a gradual decline in the faecal larvae being excreted until 3 weeks after infection. This was followed by a persistent, low grade excretion of larvae which continued until the death of all the nude mice by 45 days after infection. No larvae were seen in the faeces of the heterozygous mice after day 12. Inlestinal adult worms in primary infections Nine female C57B1/ nude mice and 12 intact controls were infected p.c. with 500 filariform larvae. Five days after infection, 4 nude mice and intact mice were killed and the small intestines removed. The remaining mice were killed and the intestine examined 42 days after infection. The results are shown in Table 1. The intact miee had significantly greater adult worm numbers than did the nude mice on the 5th day. In nude miee, however, adult worms persisted for at least weeks after infection, whereas none were seen in control mice at this time. The experiment was repeated using BALB/c nude mice. Similar trends were noted 5 days after infection, but insufficient mice survived until day 42 for assessment. TABLE 1 Adult worms and rhabditiform larval numbers in the small intestines of C57BI/ nude mice and intact mice, 5 and 42 days after infection. Fecundity was also examined. Adults Rhabditiform Fecundity nu/nu intact nu/nu intact nu/nu intact Mice/ group Day 5 (Mean ± S.D.)* 13 ± ± ± ± ±8 47 ±37 "mean larvae per g of faeces ± S.D. <0 02 < N.S. Mice/ group Day 42 (Mean ± S.D.)* ± ± ± 18 0 Fecundity in primary infection In addition to quantitating adult worms (all of which were female) in the experiment described above, the number of rhabditiform larvae in the intestine of each mouse was counted. Fecundity was estimated by dividing the number of rhabditiform larvae in each mouse by the number of adult worms in that animal. The rhabditiform larval numbers and the mean values for fecundity are shown in Table 1. While there were significantly more larvae in the intestines of intact mice than in nudes, there was no significant difference in the fecundity of adult worms in the two groups. In a repeat experi- Probability Probability <0 05 <0 001 <0 025

4 184 H. J. S. DAWKINS. G. F. MITCHELL AND D. L GROVE ment using BALB c hypothymic mice, very similar results were obtained 5 days after infection but there were insufficient anitnals for a calculation to be made on day 42. Effect of route of infection Five female BALB c nude mice were infected p.c. with S. ratti larvae and 4 were injected s.e. with the same number of larvae. Seven days later, the faecal larval excretion was measured. Significantly more larvae were seen in the mice which received a subcutaneous injection. There were 310 ± 290 (mean ± S.D.) larvae and.70 ± larvae per g of faeces (p <0.001) for mice infected p.c. and s.e. respectively. Acquisition of resistance Twelve female BALB/c nude mice were injected s.e. with 00 filariform larvae and the faecal larval excretion measured weekly. Four weeks after infection mice from this group were given a challenge infection of 850 filariform larvae as were nude control animals which had not been exposed previously. The faecal larval excretion of each of these groups was measured for a further 2 weeks. The results are expressed in Table 2. No significant differences were seen between nude mice receiving a primary infection and those given a challenge infection when examined 1 and 2 weeks after the secondary infection. DISCUSSION The duration of infection in congenitally hypothymic fnu/nu) mice after subcutaneous injection of 5. ratti has been examined by excretion of larvae in the faeces. The number of adult worms and the fecundity of those females have been compared in nude and intact mice following p.c. infection. Finally, the ability of a sensitising infection to induce resistance to re-infection in nude mice has been investigated. Infections in nude mice are dramatically altered when compared with infections in intact animals. The peak faecal larval excretion occurs at about days 12 to 14 instead of day 7 as in normal tnice. The increased excretion of larvae in the faeces 2 weeks after infection indicates either larger numbers of worms in the small intestine or greater fecundity of those worms at that time. TABLE 2 Eaccat tarvat excretion in primary aud secondary infections. There were mice in each group. Days after infection Primary infection (mean ±S,D.)t 480 ± ±.^ ± ± ± 00 Secondary Tnfected mice (mean ±S.D.)t. 000 ± ^ \ 00 * Challenge infection given 28 days after primary infection. t Mean larvae/g of faeces ± S.D. infection* Control mice (mean :l:s.d.)t 150 = t = t4700

5 STRONGYLOIDIASIS IN NUDE MICE 185 Unfortunately, we were unable to obtain further supplies of susceptible, inbred strains of mice with which to examine these points. In addition, the duration of infection is increased markedly from less than 2 weeks in control mice to at least weeks in nude miee. The increased numbers of larvae excreted in hypothymic animals is similar to the observation made in mice immunosuppressed with prednisolone (Grove and Dawkins, 1981). In contrast to the latter situation in which there was only a slight increased duration of infection, however, excretion of larvae in nude mice persisted for as long as the animals survived. A similar persistence in nude mice has been observed in a number of other intestinal parasitic infections (Mitchell, 1982) such as Nippostrongylus brasiliensis (Jaeobson and Reed, 1974). Trichinella spiralis (Ruitenberg er «/ ), Hymenolepis diminuta (Tsaak, Jaeobson and Reed, 1975) and Giardia miiris (Stevens, Frank and Mahmoud, 1978). Thus, T cell-mediated immune proces.ses play a major role in the spontaneous expulsion of 5. ratti from the intestines of mice in a primary infection. S. stercoralis has an unusual ability to replicate within the host. Moqbel and Dcnham (1978) suggested that 5. ratti may have a similar capacity in immunosuppressed rats. In contrast, however, Olson and Schiller (1978) found no evidence for autoinfeetion in rats, nor did we observe autoinfection in mice immunosuppressed with corticosteroids (Grove and Dawkins, 1981). In the present study, we found adult worms in the bowel weeks after infection. The number of worms counted at this time was considerably less than those found 5 days after infection. Thus, there is no positive evidence to indicate autoinfection, as would have been suggested if adult worm numbers had progressively risen. It seems more likely that the worms found weeks after infection in nude miee have persisted in the intestine for that period. The recovery of adult worms from the intestines in mice infected p.c. in the initial experiment was lower than that anticipated from previous experience. This suggested that the hairless skin of nude miee may have impaired the penetration of infective larvae. This was confirmed when a direct comparison on the intensity of infection was made between mice infected p.c. and those injected s.c. thus by-passing the skin barrier. This resistance may be due ei.ther to paucity of hair follicles through which they may enter (Abadie, 193) or to changes in the stratum corneum which may inhibit direct penetration of the infective larvae (Dawkins, Muir and Grove, 1981). Challenge infections of nude mice revealed that there is no acquisition of resistance to re-infection. A similar inability of nude mice to acquire resistance to re-infection has been shown in other intestinal parasitic infections including Nematospiroides duhiiis (Prowse et al ) and C. muris (Stevens et ai, 1978). The inability of nude mice to resist a secondary infection indicates the important role of T cell-dependent events in the acquisition of resistance to challenge infection with 5. ratti. The capacity to confer profound resistance in mice with relatively small numbers of mesenteric lymph node cells (Dawkins and Grove, 1981b) is further support for this view.

6 18 H. J. S. DAWKINS, G. F. MITCHELL.\ND D. I. GROVE Furthermore, the inability to acquire resistance should facilitate replication of 5. ratti within the hypothymic host. Thus, failure to find increased numbers of adult worms in the small intestine weeks after infection is additional support for the view that autoinfection does not occur in 5. ratti infections in mice. In conclusion, it has been shown that the host-parasite relationship is altered in 5. ratti infections of nude mice. Further investigations are required to delineate the precise nature of these changes. Acknowledgements. This study was supported by a grant from Ihe Rockefeller Foundation. ABADIE, S. H. (193): 'The life cycle of Strongyloidi's ratti.' J. Parasitol.. 49, 241. DAWKINS, H. J. S., and GROVE, D. I. (1981a): 'Kinellcs of primary and secondary infections with Strongyloides ratti in mice,' Int. J. Parasitol., 11, 89. DAWKINS, H. J. S., and GROVE, D. I. (1981b): 'Transfer by serum and cells of resistance to infection with Strongyloides ratti in mice.' Immunology, 43, 317. DAWKINS, H. J. S., and GROVE, D. I. (1982): 'Infection with Strongyloides stercoralis in mice and other laboratory animals.' J. HelminthoL, accepted for publicatiod, DAWKINS, H. J. S., GROVE. D. I.. DuNSMORE, J. D., and MITCHELL, G. F. (1980): 'Strongyloides ratti: susceptibility to infection and resistance to reinfection in inbred strains of mice as assessed by excretion of larvae.' Int. J. Parasitol., 10, 125. DAWKINS, H. J. S.. Mum, G. M., and GROVE. D. I. (1981): 'Histopathological appearances in primary and secondary infections with Strongyloides ratti in mice.' Int. J. Parasitol., 11, 97. GALLIARD, H. (197): 'Pathogenesis of Strongyloides.' Helminthol. Abst., 3, 247. GROVE, D. I., and DAWKINS, H. J. S. (1981): 'Effects of prednisolone on murine strongyloidiasis." Parasitology, 83,401., D. D., JACOBSON, R. H., and REED. N. D. (1975): 'Thymus dependence of tapeworm {Hymenolepis diminuta) elimination from mice.' tnfect. Immun., 12, REFERENCES JACOBSON, R. H., and REED, N. D. (1974): 'The immune response of congenitaliy athymic (nude) mice to the intestinal nemalode Nipposirongylus brasiliensis.' Proc. Soc. Exp. Biol. Med., 147, 7. MrrcHtLL, G. F. (1982): The nude mouse in immunoparasitology.' In "The Nude Mouse in Experimental and Clinical Research." Vol. II. (Eds. Fogh, J. and Giovanella. B. C.) Academic Press, New York, p. 27, accepted for publication. MoQBiiL, R.. and DENHAM. D. A. (1978); 'Strongyloides ratti: the effect of betamelhasone on the course of infection in rats." Parasitol OLSON, C. E., and SCHILLER. E. L. (1978): 'Strongyloides ratti infection in rats. II. Effects of cortisone treatment.' Am. J. Trap. Med. Hyg., 27, 527. PROWSE, S. J., MITCHELL, G. F.. EY. P. L., and JENKIN. C. R. (1978): 'Nematospiroides dubius: Susceptibility to infection and the development of resistance In hypothymic (nude) BALB/c mice.' Aust. J. Exp. Biol. Med. Sci.. 5,.S1. RUITKNBERG, E. i.. ELEGERSMA. A., KRUIZINGA, W., and LEENSTRA, F. (1977): 'Trichinella spiralis infection in congenitaliy athymic (nude) mice. Parasitological. serological and haematological studies with observations on intestinal pathology.' Immunology. 33, 581. STEvr.Ns. D. P.. FRANK, D. M., and MAHMOUD, A. A. F. (1978): 'Thymus dependency of host resistance to Giardia muris infection: Studies in nude mice.' /. Immunol.,

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