Aust. J. Exp. Biul. Med. Sci., 60 (Pt.2) 181.186(1982)

© STRONGYLOIDES RATTI INFECTIONS IN CONGENITALLYHYPOTHYMIC (NUDE) MICE

by H. J. S. DAWKINS, G. F. MITCHELL^' AND D . I. GROVE

(From the Department of Medicine, University of Western Australia, QueenElizabeth II Medical Centre, Nedlands, Western Australia 6009, and *Laboratoryof 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 incongenitally hypothymic CBA/H, C57B1/6 and BALB/c nude mice. The intensityof infection and the duration of faecal larval excretion were both increased innude mice when compared with intact mice. Adull worms persisted in the smallintestine of nude mice for at least 6 weeks. Greater worm burdens were foundin sLich mice after subcutaneous injection as compared wiih percutaneous infec-tion. Hypothymic mice did not acquire resistance to re-infection. It is concludedthat both the spontaneous expulsion of worms in primary infection and resistanceto challenge infection are T cell-dependent events. Autoinfection was not seen.

INTRODUCTIONStrongyloidiasis is one of the major human intestinal nematode infec-

tions. Unfortunately, Strongyloides stercoralis is not well adapted to smalllaboratory animals (Galliard, 1967; Dawkins and Grove, 1982). Conse-quently, the closely related species, 5. ratti, has been used in experimentalmodels of this infection. We have recently developed a murine model forstrongyloidiasis (Dawkins et al, 1980) and shown that mice acquire a pro-found resistance to re-infection (Dawkins and Grove, 1981a). Resistancecan be transferred by immune serum and cells (Dawkins and Grove, 1981b)and is partially abrogated by immunosuppression with corticosteroids (Groveand Dawkins, 1981). In this study we examine the kinetics of primary andsecondary infections with S. ratti in hypothymic mice. Thus, the role of Tcell-mediated events in the spontaneous expulsion of worms which occurs inprimary infections and in the development of resistance to challenge infec-tions is further delineated.

MATERIALS AND METHODSAnimals

Young adult female CBA/H, C57B1/6 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.

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, 150-200 g in weight, were supplied by the Animal BreedingUnit, University of Western Australia (Perth, W.A.).

Strongyloides ratti

An homogonlc Strain of S. ratti (originally obtained from Dr. P. A. G. Wilson, Universityof Edinburgh) was maintained by serial passage in SD rats. Methods for infecting mice bothpercutaneously (p.c.) and subcutaneousiy (s.c.) and for quantifying adult worms in theintestine, 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 intaetanimals were infected s.c. with 400 filariform larvae of 5. ratti. The faecesfrom these animals were examined at various intervals after infection (Fig.1).

In the control mice, the kinetics of faecal larval excretion followed thatusually seen in nonnal mice. Peak numbers of larvae were seen 6 days afterinfection. This was followed by a rapid drop in numbers, with no larvae

1 6 -

2 -

0 -

IS

TIME

20

AFTER

X 36 40

INFECTION (DAVSt

Fig. 1. Faecal larval excretion in hypothymic CBA/H (nu/nu) mice (• • ) and inintact control mice (O O) at various intervals after infection. There were 10 mice pergroup and the results are expressed as geometric mean ± S.E.M.

STRONGYLOIDIASIS IN NUDE MICE 183

being observed 12 days after infection. The faecal larval excretion of the nudemiee was not signifieantly different from that of the intact animals when exa-mined 6 days after infection. When counted on days 9 and 12, however, thefaecal larval numbers had increased two-fold (p < 0 001 in each instancewhen compared with the faecal larvae in the same animals 6 days after infec-tion). Thereafter, there was a gradual decline in the faecal larvae beingexcreted until 3 weeks after infection. This was followed by a persistent, lowgrade excretion of larvae which continued until the death of all the nude miceby 45 days after infection. No larvae were seen in the faeces of the hetero-zygous mice after day 12.

Inlestinal adult worms in primary infectionsNine female C57B1/6 nude mice and 12 intact controls were infected

p.c. with 500 filariform larvae. Five days after infection, 4 nude mice and 6intact mice were killed and the small intestines removed. The remaining micewere killed and the intestine examined 42 days after infection. The resultsare shown in Table 1. The intact miee had significantly greater adult wormnumbers than did the nude mice on the 5th day. In nude miee, however, adultworms persisted for at least 6 weeks after infection, whereas none were seenin control mice at this time. The experiment was repeated using BALB/cnude mice. Similar trends were noted 5 days after infection, but insufficientmice survived until day 42 for assessment.

Fecundity in primary infectionIn addition to quantitating adult worms (all of which were female) in

the experiment described above, the number of rhabditiform larvae in theintestine of each mouse was counted. Fecundity was estimated by dividingthe number of rhabditiform larvae in each mouse by the number of adultworms in that animal. The rhabditiform larval numbers and the mean valuesfor fecundity are shown in Table 1. While there were significantly more larvaein the intestines of intact mice than in nudes, there was no significant differ-ence in the fecundity of adult worms in the two groups. In a repeat experi-

TABLE 1Adult worms and rhabditiform larval numbers in the small intestines of C57BI/6 nude mice

and intact mice, 5 and 42 days after infection. Fecundity was also examined.

Adults nu/nu

intactRhabditiform nu/nu

intactFecundity nu/nu

intact

Mice/group

4

64

64

6

Day 5(Mean ±S.D.)*

13 ± 10

70 ±33230 ± 82

2420 ± 93013 ± 8

47 ±37

Prob-ability

< 0 02

< 0 005

N.S.

Mice/group

5

65

65

6

Day 42(Mean ±

S.D.)*

6 ± 5 - 5

0440 ± 190

022 ± 18

0

Prob-ability

< 0 05

< 0 001

< 0 025

"mean larvae per g of faeces ± S.D.

184 H. J. S. DAWKINS. G. F. MITCHELL AND D . L GROVE

ment using BALB c hypothymic mice, very similar results were obtained 5days after infection but there were insufficient anitnals for a calculation to bemade on day 42.

Effect of route of infectionFive female BALB c nude mice were infected p.c. with 1.000 S. ratti

larvae and 4 were injected s.e. with the same number of larvae. Seven dayslater, the faecal larval excretion was measured. Significantly more larvaewere seen in the mice which received a subcutaneous injection. There were310 ± 290 (mean ± S.D.) larvae and 6.670 ± 2.350 larvae per g of faeces(p <0.001) for mice infected p.c. and s.e. respectively.

Acquisition of resistanceTwelve female BALB/c nude mice were injected s.e. with 600 filariform

larvae and the faecal larval excretion measured weekly. Four weeks afterinfection 6 mice from this group were given a challenge infection of 850filariform larvae as were 6 nude control animals which had not been exposedpreviously. The faecal larval excretion of each of these groups was measuredfor a further 2 weeks. The results are expressed in Table 2. No significantdifferences were seen between nude mice receiving a primary infection andthose given a challenge infection when examined 1 and 2 weeks after thesecondary infection.

DISCUSSIONThe duration of infection in congenitally hypothymic fnu/nu) mice

after subcutaneous injection of 5. ratti has been examined by excretion oflarvae in the faeces. The number of adult worms and the fecundity of thosefemales have been compared in nude and intact mice following p.c. infection.Finally, the ability of a sensitising infection to induce resistance to re-infectionin nude mice has been investigated.

Infections in nude mice are dramatically altered when compared withinfections in intact animals. The peak faecal larval excretion occurs at aboutdays 12 to 14 instead of day 7 as in normal tnice. The increased excretion oflarvae in the faeces 2 weeks after infection indicates either larger numbers ofworms in the small intestine or greater fecundity of those worms at that time.

TABLE 2Eaccat tarvat excretion in primary aud secondary infections. There were 6 mice in each group.

Days afterinfection

71421283542

Primary infection(mean ±S,D.) t

480 ± 400690 ± .̂ 30300 ± 670170 ±380300 ± 600

—

SecondaryTnfected mice

(mean ±S.D. ) t.————

6000 ± 60007800 ^ \ 600

infection*Control mice

(mean :l

1650 =4080 =

:S.D.)t

t 1600t4700

* Challenge infection given 28 days after primary infection.t Mean larvae/g of faeces ± S.D.

STRONGYLOIDIASIS IN NUDE MICE 185

Unfortunately, we were unable to obtain further supplies of susceptible, in-bred strains of mice with which to examine these points. In addition, theduration of infection is increased markedly from less than 2 weeks in controlmice to at least 6 weeks in nude miee.

The increased numbers of larvae excreted in hypothymic animals issimilar to the observation made in mice immunosuppressed with prednisolone(Grove and Dawkins, 1981). In contrast to the latter situation in which therewas only a slight increased duration of infection, however, excretion of larvaein nude mice persisted for as long as the animals survived. A similar persis-tence in nude mice has been observed in a number of other intestinal parasiticinfections (Mitchell, 1982) such as Nippostrongylus brasiliensis (Jaeobsonand Reed, 1974). Trichinella spiralis (Ruitenberg er «/.. 1977), Hymenolepisdiminuta (Tsaak, Jaeobson and Reed, 1975) and Giardia miiris (Stevens,Frank and Mahmoud, 1978). Thus, T cell-mediated immune proces.ses playa major role in the spontaneous expulsion of 5. ratti from the intestines ofmice in a primary infection.

S. stercoralis has an unusual ability to replicate within the host. Moqbeland Dcnham (1978) suggested that 5. ratti may have a similar capacity inimmunosuppressed rats. In contrast, however, Olson and Schiller (1978)found no evidence for autoinfeetion in rats, nor did we observe autoinfectionin mice immunosuppressed with corticosteroids (Grove and Dawkins, 1981).In the present study, we found adult worms in the bowel 6 weeks after infec-tion. The number of worms counted at this time was considerably less thanthose found 5 days after infection. Thus, there is no positive evidence toindicate autoinfection, as would have been suggested if adult worm numbershad progressively risen. It seems more likely that the worms found 6 weeksafter 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. inthe initial experiment was lower than that anticipated from previous experi-ence. This suggested that the hairless skin of nude miee may have impairedthe penetration of infective larvae. This was confirmed when a direct com-parison on the intensity of infection was made between mice infected p.c. andthose injected s.c. thus by-passing the skin barrier. This resistance may bedue ei.ther to paucity of hair follicles through which they may enter (Abadie,1963) or to changes in the stratum corneum which may inhibit direct pene-tration of the infective larvae (Dawkins, Muir and Grove, 1981).

Challenge infections of nude mice revealed that there is no acquisitionof resistance to re-infection. A similar inability of nude mice to acquire resist-ance to re-infection has been shown in other intestinal parasitic infectionsincluding Nematospiroides duhiiis (Prowse et al.. 1978) and C. muris(Stevens et ai, 1978). The inability of nude mice to resist a secondary infec-tion indicates the important role of T cell-dependent events in the acquisitionof resistance to challenge infection with 5. ratti. The capacity to confer pro-found resistance in mice with relatively small numbers of mesenteric lymphnode cells (Dawkins and Grove, 1981b) is further support for this view.

186 H. J. S. DAWKINS, G. F. MITCHELL .\ND D . I. GROVE

Furthermore, the inability to acquire resistance should facilitate replicationof 5. ratti within the hypothymic host. Thus, failure to find increased numbersof adult worms in the small intestine 6 weeks after infection is additionalsupport for the view that autoinfection does not occur in 5. ratti infections inmice.

In conclusion, it has been shown that the host-parasite relationship isaltered in 5. ratti infections of nude mice. Further investigations are requiredto delineate the precise nature of these changes.

Acknowledgements. This study was supported by a grant from Ihe RockefellerFoundation.

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