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Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines
EXPERIMENTAL
PARASITOLOGY
Heligmosomoides
52, 69-76 (1981)
polygyrus: Simple Recovery Larvae from Mouse Intestines
PETER L. EY, STEPHEN J. PROWSE,' Depurtmmt
of Microbiology
AND CHARLES R. JENKIN
und Immunology, The University Adelaide, 5001, Austruliu (Accepted
for publication
of Post-Infective
of Adeluidc,
G.P.O. Box 498,
13 May 1980)
C. R. 1981. HeliRmosomoidespolygyrus: Simple EY, P. L., Paowst, S. J., AND J~NKIN, recovery of post-infective larvae from mouse intestines. Experimental Purusitology St, 69-76. Mice infected orally with third-stage larvae of HeliXmosornoides po/ygyru.s were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hrinfected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36-72 hr before necropsy. INDEX DESCRIPTORS: Heligmnsomoides polygyrus (= Nemutospiroides duhius); Nematode, parasitic; Mouse; Larval recovery; lntestinal development.
IINTRODUCTION Infection of mice by the gastrointestinal nematode Heligmosomoides polygyrus (=Nematospiroides dubius) has been studied in a number of laboratories, particularly with regard to the immunopathological changes which occur in infected animals and the development of protective immunity (Van Zandt 1961; Liu 1965; Panter 1969; Bartlett and Ball 1974; Crandall et al. 1974; Cypess 1974; Jones and Rubin 1974; Dobson and Owen 1977). Mice may be effectively immunized against reinfection by giving live third-stage (infective) larvae by the natural oral route or by intravenous, intraperitoneal, or subcutaneous injection (Chaicumpa et al. 1977). Living third-stage larvae always penetrate the wall of the intestine, even in mice which are highly immune and regardless of the route used for im’ Present address: Department of Immunology, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, 2601, Australia.
munization. Immunity is invariably directed against the third-stage larvae following their penetration of the wall of the small intestine but not (in the outbred strain of mice we have used) against the adult worm (Chaicumpa et al. 1977; Prowse et al. 1979). The critical period for induction of immunity (measured by the number of encysted larvae present in the intestinal wall 5 days postchallenge (p.c.) or of adult worms present in the lumen of the intestine 210 days p.c.) seems to be during the initial 5 or 6 days of an infection since animals infected with third-stage larvae and then purged daily with Pyrantel on Days 6-10 postinfection (p.i.) (to eliminate adult worms as they return to the lumen of the intestine) show a significant degree of resistance to reinfection. In contrast, immunity is not induced in mice given adult worms by stomach tube transfer, although the worms can be carried for considerable periods of time (unpublished results). These findings suggest that the presence 69 00144894/81/040069-08$02.00/O Copyright AU rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
70
EY, PROWSE, AND JENKIN
of particular developmental stages of H. polygyrus larvae in the lamina propria and muscularis of the intestine is important for the induction of protective immunity. It has proved extremely difficult, however, to recover larvae from the intestines of mice during this period of infection and this has complicated studies on both the development of the early parasitic stages and the nature of the immunologically important substances that they synthesize and perhaps secrete. The experiments described below demonstrate that viable larvae suitable for such studies can be recovered easily and with little contamination from infected mice at any time from 1 to 7 days p.i., at which time the parasites begin to migrate back into the lumen of the intestine. MATERIALS
AND METHODS
Host animal. Outbred 6-week-old LACA mice of either sex, weighing about 20 g, were obtained from a closed, specific pathogen-free colony maintained at the University animal facility. The mice were provided with unlimited food and water and conventionalized for several weeks before use. Parasite. Third-stage infective larvae of Heligmosomoides polygyrus were supplied originally by Dr. R. I. Sommerville, Zoology Department, University of Adelaide. The parasite was maintained by culturing the feces of infected mice on moist filter paper at room temperature (20-25 C). After 7 days of culture, third-stage larvae were collected, washed in distilled water, and stored at 4 C. The procedure has been described in detail by Chaicumpa et al. (1977). Infection of mice. Third-stage larvae, suspended in 0.4 ml of distilled water, were introduced into the lower part of the esophagus using a blunt 19-gauge needle connected to a tuberculin syringe. The number of larvae given was varied and is indicated in conjunction with the results. Collection of post-infective emergent lar-
vae. Mice were killed by cervical dislocation at selected times after infection. The entire length of the small intestine from just above the pyloric sphincter to the ileocecal valve was immediately removed and freed of most connective tissue, taking special care not to cut or tear the intestinal wall. After tying the ends with surgical thread (to prevent extrusion of the lumen contents), each intestine was immersed in 15 ml of prewarmed (37 C) medium in a 50 x 13-mm plastic Petri dish (Disposable Products Pty. Ltd., South Australia) and incubated at 37 C for the period indicated. The medium was a 1:4 mixture of Medium 199 (Commonwealth Serum Laboratories, Melbourne, Australia) and physiological saline and contained 200 IU/ml of both penicillin and streptomycin sulfate. At the end of the incubation period, the intestines were removed from the dishes and discarded. Larvae which had emerged through the wall of the intestine into the medium were inspected under a dissecting microscope and counted as they were removed using a Pasteur pipet. Transfer of emergent larvae or adult worms. Mice were infected with 600 thirdstage larvae and killed at selected times thereafter. The intestines were incubated at 37 C for 45 min. Larvae which emerged during this period were immediately dispensed in groups of 150 into small glass tubes. The worms, which settled on the bottom of the tubes, were sucked into a tuberculin syringe and administered orally in a volume of 0.5 ml to normal recipient mice. Adult worms were collected from mice which had been infected for 12 days. The intestines were slit longitudinally, placed in warm physiological saline and the worms which moved away from the intestines were collected using a Pasteur pipet. They were transferred to recipient mice in the manner described above for emergent larvae. Recipient mice were killed 3 days after transfer (for counting encysted parasites) or 10 days after transfer, when the
Heligmosomoides
polygyrus:
number of adult worms present in the lumen was assessed. Enumeration of encysted larvae. The number of encysted larvae developing within the wall of the small intestine of mice infected with third-stage or emergent larvae was determined on Day 5 or 3 p.i., respectively (Chaicumpa et al. 1977). The small intestine was removed from just below the pyloric sphincter to the ileocecal valve and pressed between two pieces of transparent Perspex. The whole of the exposed intestine was scanned under a dissecting microscope and the cysts were counted. Immunization. Mice were infected two or three times with 20 or 200 third-stage larvae in order to induce different levels of resistance to reinfection. The larvae used for the immunizing infections were administered intravenously (Chaicumpa et al. 1977). The degree of resistance against an oral challenge with third-stage larvae was determined from the difference in the number of encysted larvae observed on Day 5 p.c. in the small intestines of preinfected compared with control mice (Table III). This difference represents the number of larvae which penetrate the wall of the intestine in immune mice but whose subsequent growth and development is prevented (Chaicumpa et al. 1977; Prowse et al. 1978, 1979; Behnke and Parish 1979). RESULTS Recovery of Emergent Larvae at Different Times after Infection
Mice, in groups of 10, were each infected with 600 third-stage Heligmosomoides polygyrus larvae and killed at various times thereafter. Their intestines were removed and incubated to recover emergent larvae. The results (Fig. 1) showed that larvae could be recovered from the intestines of mice which had been infected for a period of 18-24 hr up to 7-8 days. The intestines taken from mice infected for 40-100 hr yielded the highest numbers of larvae (approximately 18% of the infective dose).
RECOVERY FROM MICE
0
2
4
71
6
8
TIM AFTER,NFECT,M bws)
FIG. 1. Recovery of emergent larvae from the intestines of mice killed at various times after infection polygyrus. Each point reprewith Heligmosomoidrs sents the mean number of larvae recovered per intestine (IO mice per group) after a 2-hr incubation. The mice were each infected with 600 third-stage larvae and killed at the indicated times. Vertical bars represent 2 SE.
Since 18-24 hr must elapse before all the larvae given in an oral dose have penetrated the intestinal epithelium and most of the worms have returned to the lumen of the intestine by Day 8 of infection (Bryant 1973), this period of maximal recovery corresponds to the time during which the larvae reside in the lamina propria and muscularis. The development (including molts) of the parasites could be observed by examining the emergent larvae recovered after different periods of infection. As is evident from Fig. 2, the 36- to 120-hr period was characterized by rapid growth of the larvae accompanied by sexual maturation which was usually evident by 50-60 hr p.i. The larvae (of either sex) recovered from any single animal varied considerably in size (approximately fivefold), possibly reflecting differences in growth rate and/or differences in the time at which the larvae exsheathed and penetrated the mucosal epithelium. Recovery of Emergent Larvae: Relationship to Dose
To determine whether the emergence of larvae was due to an “overcrowding” effect in the intestinal wall, mice were given various doses of third-stage larvae ranging
72
EY, PROWSE,
AND JENKIN
FIG. 2. Emergent larvae obtained from the intestines of mice infected with He/i~/no.\omoides po/yg.~?‘vus (600 third-stage larvae per mouse) and killed at the indicated times after infection. (A) Third-stage (pre)infective larvae. (B) 36, (C) 48. (D) 72, (E) 96, (F) 120, (G) 144, and (H) 168 hr. Magnification, 18x.
in number from 100 to 2400 larvae per mouse. The animals were killed 48 hr after infection and the number of larvae emerging from each intestine during a 2-hr incubation was determined. The results (Table I) demonstrated that the size of the infective dose determines the number of larvae which emerge from the isolated intestines. It is clear, however, that TABLE 1 Recovery of Emergent Larvae of Hr/ijimoJo/,loidf,,~ po/yg.~rrr.c: Relationship to Infective Dose Larvae
Dose” 100 300 600 1200 1800 2400
Mean number ? 12.7 36.2 99.0 184.6 355.3 414.0
2 + k I 2 i
4.4 11.3 20.9 32.9 60.8 80.5
recovered SE”
(48 hr pi.) Percentage 12.7 12.1 16.5 15.4 19.7 17.2
of dose k ii i 2 _t
4.4 3.8 3.5 2.1 3.4 3.3
” Mice (IO per group) were each infected with the indicated number of third-stage larvae. After 48 hr. the animals were killed and the number of larvae emerging from each intestine during a t-hr incubation was determined. Ir The standard error (SE) of each mean is shown.
the recovery of larvae, taken as a proportion of the infective dose, was similar over the entire dose range examined. The mean percentage recovery (+ 1 SEM) over this range was 15.6 ? 1.2. The differences of the means for each dose group from this value and from each other were insignificant. It therefore seems highly unlikely that the emergence of larvae is a phenomenon associated with “overcrowding.” This conclusion is supported by the observation that no larvae were present in the peritoneal exudates collected just before the intestines were removed from mice given up to 10,000 larvae 48 hr previously. Rate of Emergence during Incubution In order to determine the rate at which larvae emerge from the intestines during incubation, the intestines of mice killed 48 hr after receiving 600 third-stage larvae were incubated for either 3 hr or for three consecutive 40-min periods, between which they were transferred to fresh medium. The
Heligmosomoides
polygyrus:
results of this experiment (Table II) showed that of the larvae recovered over a 2- to 3-hr period, 80% emerged during the first 40 min, 11% during the next 40 min, and 9% in the 80- to 120-min period.
TABLE II Recovery of Emergent Larvae of Hrligmosomoidrs polygyrus from Intestines Incubated in Vitro for Different Lengths of Time Period of incubation” (min) O-40 40-80 80- 120 O-180
Recovery of Larvae from Immunized Mice
Previous studies have established that mice which exhibit resistance to reinfection with H. pofygyrus are unable to prevent the penetration of the mucosal epithelium of the small intestine by infective larvae (Chaicumpa et al. 1977; Prowse et al. 1979; Behnke and Parish 1979). Following penetration, some but not all of the larvae are arrested in their growth and development before they reach a size which is discernable in the intestinal tissue under a dissecting microscope (3.5-4 days if not arrested). These larvae are eventually killed within the granulomatous lesions which develop (Bartlett and Ball 1974; Jones and Rubin 1974; Chaicumpa et al. 1977; Prowse et al. 1979). No data are available, however, to indicate how quickly the larvae are attacked and damaged after penetrating the mucosal epithelium. In the hope of obtaining such information, the number of larvae emerging from the intestines of preinfected and normal mice killed 36, 48, or 72 hr after receiving an oral challenge of third-stage larvae was determined. The results of three experiments are presented in Table III. It can be seen that significantly fewer larvae emerged from the intestines of immunized mice than from those of control mice. Moreover, there seemed to be some correlation between the degree of immunity expressed by the mice and the number of emergent larvae recovered. In Expt 1, for example, mice immunized with two or three minor infections (20 larvae per dose) showed a small reduction (19 and 29%, respectively) in the number of Day 5 cysts relative to the control value, and a slightly larger reduction (47 and 62%, respectively) in the number of
73
RECOVERY FROM MICE
Number of larvae recovered (mean ? SE) 87.6 k 12.3 5 9.8 k 100.7 +
20.3 1.4 1.1 16.3
” Twenty mice were each infected with 600 thirdstage larvae and killed after 48 hr. Half of the intestines were subjected to three consecutive 40-min incubations; they were transferred to new Petri dishes for each incubation. The remaining 10 intestines were given a single incubation (180 min).
48-hr p.c. emergent larvae. Immunization with a large number of third-stage larvae (three doses of 200 larvae; Chaicumpa et al. (1977); Prowse et al. (1978)) resulted in a greater degree of immunity (Expt 2, 78%) and a very marked reduction (>90%) in the number of larvae emerging from the isolated intestines (Expts 2 and 3). Viability
of Emergent Larvae
Providing the intestines were not incubated for an excessively long period (>60-90 min), the larvae recovered in the medium were intact and motile. To test their viability, larvae collected from the intestines of mice infected 48 hr previously were incubated at 37 C for several days in Medium 199 containing 10% fetal calf serum and 200 IU/ml of both penicillin and streptomycin sulfate (200 larvae in 15 ml of medium; loo-ml bottles, flat face down). During this period, the parasites continued to grow and differentiate. At least one molt was observed, the worms appeared to develop sexually and most were still alive after 6 days incubation (results not shown). The infective potential of emergent larvae was investigated by collecting larvae from the intestines of mice infected for different periods of time and administering them by stomach tube to normal mice. When the adoptive hosts were killed 10
74
EY, PROWSE,
AND JENKIN
TABLE III of Emergent Larvae of Hrligmosomoidrs po/q’g.y:.,‘rusfrom the Intestines of Normal and Preinfected Mice after an Oral Challenge with Third-Stage Larvae
Recovery
Experiment I”
Previous administrations of L:,
Number of emergent larvae” (hr postchallenge) Number of cysts” (Day 5 postchallenge)
None 2 x 20 3 x 20
210 2 3.3 171 + IO 150 + 13
2’
None 3 x 200
200 +- 9.0 43.2 k 7.4
3”
None 3 x 200
36 109 k I3 2.3 t 2.9
48 108 i 25 57 t 10 41 + IO 99 k 21 4.9 2 I.1 109 -t I6 3.4 -c I.5
72 119 k 15 15.1 t 4.6 -
” Mean ? SE (10 mice per group). Emergent larvae were collected over a 2-hr incubation. b Immunized mice were injected intravenously (iv) with 20 third-stage larvae (L:,) on Days -37 (3x infected only), -30, and - 16. All mice, including the controls. were purged with 3 mg of Pyrantel pamoate (Pfizer) on Days -8 and -6. Each was orally challenged on Day 0 with either 200 L:, (for cyst counts) or 600 L:, (for larval recovery). ’ Immunized mice received 200 L:, iv on Days -88, -56. and -40. All were given Pyrantel on Days -10 and -7 and challenged on Day 0 as in footnote b. ” Immunized mice received 200 L., iv on Days -53. -38, and -21. They were challenged on Day 0 as in footnote b.
days later, a proportion (1 l-36%) of the transferred parasites were found in the lumen of the small intestine as (apparently) mature adult worms (Table IV). The observed recoveries can be considered as minimal values of survival, since some of
TABLE IV Ability of Emergent Larvae of H~ligmosomoidr.~ polygyrus Collected at Different Times after Infection to Continue Development upon Oral Transfer to a Second Host Time after infection at which larvae were collected for transfer” (hr) 48 h 72 h 96 h 120 h 144 h Adult worms
Number’, of parasites present in the second host Day 3 (cysts) 15.2 + 3.5
Day IO (adults)
-
17.4 53.9 51.2 37.3 51.5
-
I I.5 f 3.9
’ I50 larvae or adult worms per mouse. Ir Mean I SE (five mice per group).
k + k 2 +
3.6 6.8 12.7 7.7 25.7
the transferred larvae may have been damaged during ejection through the syringe needle and passage through the stomach. The possibility of injury is exemplified by the very low survival of adult worms (used as a control) following similar adoptive transfer. These worms are adapted for existence within the lumen of the intestine, but they are much larger and they became entangled and blocked the needle during transfer. As a result, many must have been injured. DISCUSSION
The
cycle of Heligmosomoides has been studied in various detail by a number of investigators (Spurlock 1943; Baker 1954; Ehrenford 1954; Fahmy 1956; Dobson 1960), most recently by Bryant (1973) who has comprehensively reviewed the earlier data. After ingestion by the mouse, infective third-stage larvae exsheath in the stomach (Sommerville and Bailey 1973) and small intestine (Bryant 1973) before penetrating the mucosal epi-
polygyrus
life
Heligmosomoides
polygyrus:
thelium of the small intestine. By 24 hr, no larvae can be found in the lumen (Bryant 1973; Ey, unpublished data), indicating that most of them have penetrated the mucosa by this time. The larvae molt at approximately 90 hr p.i. (third to fourth stage) and 150 hr (fourth to fifth adult stage) and most of the worms return to the intestinal lumen by 190 hr to adopt their usual adult position (Bryant 1973). One of the limiting factors in investigations on the early parasitic stages of H. polygyrus has been the difficulty in excising individual larvae from the wall of the intestines of infected mice in numbers sufficient for study (e.g., Bryant 1973, 1974; Dennis 1976). This is a particular problem when one requires hundreds, if not thousands, of larvae for collecting secretions or extracts for immunochemical work. Attempts to culture the parasite in vitro have met with only partial success (Jones and Weinstein 1957; Sommerville and Weinstein 1964; Yasuraoka and Weinstein 1969). The results presented in this paper demonstrate that large numbers of larvae in the early parasitic stages of the life cycle can be recovered from infected mice with little effort. With a small amount of care, they can be collected virtually free from contamination with intestinal matter. The period for maximal recovery of larvae is between 40 and 100 hr p.i., although reasonable yields may be obtained at any time up to 6 or 7 days p.i. when the parasites begin to migrate back to the lumen. Most if not all of the emergent larvae collected at different times after infection were viable and many were able to continue their development under suitable conditions both in vitro and in vivo. At least some of the surviving parasites must penetrate the intestinal mucosa of the adoptive host in order to continue development, since encysted larvae were observed in the intestines of mice given 48-hr p.i. emergent larvae 3 days previously (Table IV). Whether or not the more developed larvae (e.g.,
RECOVERY FROM MICE
75
those collected 96-120 hr p.i.) must also penetrate the intestinal wall of an adoptive host to continue their development remains to be determined. The number of emergent larvae recovered from isolated intestines constituted a constant proportion of the dose of larvae given to the mice over a wide dose range (100-2400). Mice infected with up to 10,000 third-stage larvae have yielded a similar proportion of emergent larvae, although these mice do not survive more than a few days. Most of the larvae emerge from the intestines during the initial 40-60 min of incubation at 37 C. The phenomenon does not occur within the mouse but is apparently induced upon the death of the animal and isolation of the intestine. It is vital to commence the incubation as quickly as possible after killing the mouse and to restrict the incubation period to less than 1 hr if maximal recovery and viability is desired. Peristalsis usually continues for some time during the incubation and it is necessary to ligate both ends of the intestine to prevent contamination of the medium with digested matter. The results using immunized mice indicate that infective larvae must be arrested or immobilized soon after penetrating the mucosal lining of the small intestine. This was shown by the reduced number of larvae which emerged from the intestines of immunized compared with normal mice killed as early as 36 hr after challenge. Previous work from this laboratory has shown that the ability of infective larvae to penetrate the intestinal wall is unimpaired in immune mice (Chaicumpa et al. 1977). The reduction seems to bear some relationship to the degree of resistance expressed by the mice, i.e., to the proportion of larvae in a challenge dose which are killed by the mouse. A considerable period of time (l-3 weeks) is apparently required for the larvae to be killed, however, since immune mice treated with cortisone (an immunosuppressant) as late as lo-15 days after challenge exhibit
76
EY, PROWSE,
significantly increased worm burdens (Behnke and Parish 1979). The mechanisms responsible in immune mice for the developmental arrest, immobilization, and subsequent death of the Heligmasomoides pofygyrus larvae remain unclear. ACKNOWLEDGMENTS We are grateful to Mr. G. Ekserdjian and Ms. Ann Hallett for invaluable technical assistance, and to Dr. R. 1. Sommerville for allowing us to use his microscopes, for his interest in this work, and for helpful discussions. This work was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES
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CRANDALL, R. B., CRANDALL. C. A.. AND FRANCE. J. A. 1974. Heligmosomoides polygyrus (= Nrmutospiroides duhius). Humoral and intestinal immunologic responses to infection in mice. Exprrimentul
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S. K. 1965. Pathology of Nemuto~piroides duhius. I. Primary infections in C3H and Webster Ptrrusitology 17, 123- 135. mice. Experimrnful PANTER. H. C. 1969. The mechanism of immunity of mice to Nemutospiroide., duhius. Journul OJ 55, 38-43.
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PARASITOLOGY
Heligmosomoides
52, 69-76 (1981)
polygyrus: Simple Recovery Larvae from Mouse Intestines
PETER L. EY, STEPHEN J. PROWSE,' Depurtmmt
of Microbiology
AND CHARLES R. JENKIN
und Immunology, The University Adelaide, 5001, Austruliu (Accepted
for publication
of Post-Infective
of Adeluidc,
G.P.O. Box 498,
13 May 1980)
C. R. 1981. HeliRmosomoidespolygyrus: Simple EY, P. L., Paowst, S. J., AND J~NKIN, recovery of post-infective larvae from mouse intestines. Experimental Purusitology St, 69-76. Mice infected orally with third-stage larvae of HeliXmosornoides po/ygyru.s were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hrinfected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36-72 hr before necropsy. INDEX DESCRIPTORS: Heligmnsomoides polygyrus (= Nemutospiroides duhius); Nematode, parasitic; Mouse; Larval recovery; lntestinal development.
IINTRODUCTION Infection of mice by the gastrointestinal nematode Heligmosomoides polygyrus (=Nematospiroides dubius) has been studied in a number of laboratories, particularly with regard to the immunopathological changes which occur in infected animals and the development of protective immunity (Van Zandt 1961; Liu 1965; Panter 1969; Bartlett and Ball 1974; Crandall et al. 1974; Cypess 1974; Jones and Rubin 1974; Dobson and Owen 1977). Mice may be effectively immunized against reinfection by giving live third-stage (infective) larvae by the natural oral route or by intravenous, intraperitoneal, or subcutaneous injection (Chaicumpa et al. 1977). Living third-stage larvae always penetrate the wall of the intestine, even in mice which are highly immune and regardless of the route used for im’ Present address: Department of Immunology, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, 2601, Australia.
munization. Immunity is invariably directed against the third-stage larvae following their penetration of the wall of the small intestine but not (in the outbred strain of mice we have used) against the adult worm (Chaicumpa et al. 1977; Prowse et al. 1979). The critical period for induction of immunity (measured by the number of encysted larvae present in the intestinal wall 5 days postchallenge (p.c.) or of adult worms present in the lumen of the intestine 210 days p.c.) seems to be during the initial 5 or 6 days of an infection since animals infected with third-stage larvae and then purged daily with Pyrantel on Days 6-10 postinfection (p.i.) (to eliminate adult worms as they return to the lumen of the intestine) show a significant degree of resistance to reinfection. In contrast, immunity is not induced in mice given adult worms by stomach tube transfer, although the worms can be carried for considerable periods of time (unpublished results). These findings suggest that the presence 69 00144894/81/040069-08$02.00/O Copyright AU rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
70
EY, PROWSE, AND JENKIN
of particular developmental stages of H. polygyrus larvae in the lamina propria and muscularis of the intestine is important for the induction of protective immunity. It has proved extremely difficult, however, to recover larvae from the intestines of mice during this period of infection and this has complicated studies on both the development of the early parasitic stages and the nature of the immunologically important substances that they synthesize and perhaps secrete. The experiments described below demonstrate that viable larvae suitable for such studies can be recovered easily and with little contamination from infected mice at any time from 1 to 7 days p.i., at which time the parasites begin to migrate back into the lumen of the intestine. MATERIALS
AND METHODS
Host animal. Outbred 6-week-old LACA mice of either sex, weighing about 20 g, were obtained from a closed, specific pathogen-free colony maintained at the University animal facility. The mice were provided with unlimited food and water and conventionalized for several weeks before use. Parasite. Third-stage infective larvae of Heligmosomoides polygyrus were supplied originally by Dr. R. I. Sommerville, Zoology Department, University of Adelaide. The parasite was maintained by culturing the feces of infected mice on moist filter paper at room temperature (20-25 C). After 7 days of culture, third-stage larvae were collected, washed in distilled water, and stored at 4 C. The procedure has been described in detail by Chaicumpa et al. (1977). Infection of mice. Third-stage larvae, suspended in 0.4 ml of distilled water, were introduced into the lower part of the esophagus using a blunt 19-gauge needle connected to a tuberculin syringe. The number of larvae given was varied and is indicated in conjunction with the results. Collection of post-infective emergent lar-
vae. Mice were killed by cervical dislocation at selected times after infection. The entire length of the small intestine from just above the pyloric sphincter to the ileocecal valve was immediately removed and freed of most connective tissue, taking special care not to cut or tear the intestinal wall. After tying the ends with surgical thread (to prevent extrusion of the lumen contents), each intestine was immersed in 15 ml of prewarmed (37 C) medium in a 50 x 13-mm plastic Petri dish (Disposable Products Pty. Ltd., South Australia) and incubated at 37 C for the period indicated. The medium was a 1:4 mixture of Medium 199 (Commonwealth Serum Laboratories, Melbourne, Australia) and physiological saline and contained 200 IU/ml of both penicillin and streptomycin sulfate. At the end of the incubation period, the intestines were removed from the dishes and discarded. Larvae which had emerged through the wall of the intestine into the medium were inspected under a dissecting microscope and counted as they were removed using a Pasteur pipet. Transfer of emergent larvae or adult worms. Mice were infected with 600 thirdstage larvae and killed at selected times thereafter. The intestines were incubated at 37 C for 45 min. Larvae which emerged during this period were immediately dispensed in groups of 150 into small glass tubes. The worms, which settled on the bottom of the tubes, were sucked into a tuberculin syringe and administered orally in a volume of 0.5 ml to normal recipient mice. Adult worms were collected from mice which had been infected for 12 days. The intestines were slit longitudinally, placed in warm physiological saline and the worms which moved away from the intestines were collected using a Pasteur pipet. They were transferred to recipient mice in the manner described above for emergent larvae. Recipient mice were killed 3 days after transfer (for counting encysted parasites) or 10 days after transfer, when the
Heligmosomoides
polygyrus:
number of adult worms present in the lumen was assessed. Enumeration of encysted larvae. The number of encysted larvae developing within the wall of the small intestine of mice infected with third-stage or emergent larvae was determined on Day 5 or 3 p.i., respectively (Chaicumpa et al. 1977). The small intestine was removed from just below the pyloric sphincter to the ileocecal valve and pressed between two pieces of transparent Perspex. The whole of the exposed intestine was scanned under a dissecting microscope and the cysts were counted. Immunization. Mice were infected two or three times with 20 or 200 third-stage larvae in order to induce different levels of resistance to reinfection. The larvae used for the immunizing infections were administered intravenously (Chaicumpa et al. 1977). The degree of resistance against an oral challenge with third-stage larvae was determined from the difference in the number of encysted larvae observed on Day 5 p.c. in the small intestines of preinfected compared with control mice (Table III). This difference represents the number of larvae which penetrate the wall of the intestine in immune mice but whose subsequent growth and development is prevented (Chaicumpa et al. 1977; Prowse et al. 1978, 1979; Behnke and Parish 1979). RESULTS Recovery of Emergent Larvae at Different Times after Infection
Mice, in groups of 10, were each infected with 600 third-stage Heligmosomoides polygyrus larvae and killed at various times thereafter. Their intestines were removed and incubated to recover emergent larvae. The results (Fig. 1) showed that larvae could be recovered from the intestines of mice which had been infected for a period of 18-24 hr up to 7-8 days. The intestines taken from mice infected for 40-100 hr yielded the highest numbers of larvae (approximately 18% of the infective dose).
RECOVERY FROM MICE
0
2
4
71
6
8
TIM AFTER,NFECT,M bws)
FIG. 1. Recovery of emergent larvae from the intestines of mice killed at various times after infection polygyrus. Each point reprewith Heligmosomoidrs sents the mean number of larvae recovered per intestine (IO mice per group) after a 2-hr incubation. The mice were each infected with 600 third-stage larvae and killed at the indicated times. Vertical bars represent 2 SE.
Since 18-24 hr must elapse before all the larvae given in an oral dose have penetrated the intestinal epithelium and most of the worms have returned to the lumen of the intestine by Day 8 of infection (Bryant 1973), this period of maximal recovery corresponds to the time during which the larvae reside in the lamina propria and muscularis. The development (including molts) of the parasites could be observed by examining the emergent larvae recovered after different periods of infection. As is evident from Fig. 2, the 36- to 120-hr period was characterized by rapid growth of the larvae accompanied by sexual maturation which was usually evident by 50-60 hr p.i. The larvae (of either sex) recovered from any single animal varied considerably in size (approximately fivefold), possibly reflecting differences in growth rate and/or differences in the time at which the larvae exsheathed and penetrated the mucosal epithelium. Recovery of Emergent Larvae: Relationship to Dose
To determine whether the emergence of larvae was due to an “overcrowding” effect in the intestinal wall, mice were given various doses of third-stage larvae ranging
72
EY, PROWSE,
AND JENKIN
FIG. 2. Emergent larvae obtained from the intestines of mice infected with He/i~/no.\omoides po/yg.~?‘vus (600 third-stage larvae per mouse) and killed at the indicated times after infection. (A) Third-stage (pre)infective larvae. (B) 36, (C) 48. (D) 72, (E) 96, (F) 120, (G) 144, and (H) 168 hr. Magnification, 18x.
in number from 100 to 2400 larvae per mouse. The animals were killed 48 hr after infection and the number of larvae emerging from each intestine during a 2-hr incubation was determined. The results (Table I) demonstrated that the size of the infective dose determines the number of larvae which emerge from the isolated intestines. It is clear, however, that TABLE 1 Recovery of Emergent Larvae of Hr/ijimoJo/,loidf,,~ po/yg.~rrr.c: Relationship to Infective Dose Larvae
Dose” 100 300 600 1200 1800 2400
Mean number ? 12.7 36.2 99.0 184.6 355.3 414.0
2 + k I 2 i
4.4 11.3 20.9 32.9 60.8 80.5
recovered SE”
(48 hr pi.) Percentage 12.7 12.1 16.5 15.4 19.7 17.2
of dose k ii i 2 _t
4.4 3.8 3.5 2.1 3.4 3.3
” Mice (IO per group) were each infected with the indicated number of third-stage larvae. After 48 hr. the animals were killed and the number of larvae emerging from each intestine during a t-hr incubation was determined. Ir The standard error (SE) of each mean is shown.
the recovery of larvae, taken as a proportion of the infective dose, was similar over the entire dose range examined. The mean percentage recovery (+ 1 SEM) over this range was 15.6 ? 1.2. The differences of the means for each dose group from this value and from each other were insignificant. It therefore seems highly unlikely that the emergence of larvae is a phenomenon associated with “overcrowding.” This conclusion is supported by the observation that no larvae were present in the peritoneal exudates collected just before the intestines were removed from mice given up to 10,000 larvae 48 hr previously. Rate of Emergence during Incubution In order to determine the rate at which larvae emerge from the intestines during incubation, the intestines of mice killed 48 hr after receiving 600 third-stage larvae were incubated for either 3 hr or for three consecutive 40-min periods, between which they were transferred to fresh medium. The
Heligmosomoides
polygyrus:
results of this experiment (Table II) showed that of the larvae recovered over a 2- to 3-hr period, 80% emerged during the first 40 min, 11% during the next 40 min, and 9% in the 80- to 120-min period.
TABLE II Recovery of Emergent Larvae of Hrligmosomoidrs polygyrus from Intestines Incubated in Vitro for Different Lengths of Time Period of incubation” (min) O-40 40-80 80- 120 O-180
Recovery of Larvae from Immunized Mice
Previous studies have established that mice which exhibit resistance to reinfection with H. pofygyrus are unable to prevent the penetration of the mucosal epithelium of the small intestine by infective larvae (Chaicumpa et al. 1977; Prowse et al. 1979; Behnke and Parish 1979). Following penetration, some but not all of the larvae are arrested in their growth and development before they reach a size which is discernable in the intestinal tissue under a dissecting microscope (3.5-4 days if not arrested). These larvae are eventually killed within the granulomatous lesions which develop (Bartlett and Ball 1974; Jones and Rubin 1974; Chaicumpa et al. 1977; Prowse et al. 1979). No data are available, however, to indicate how quickly the larvae are attacked and damaged after penetrating the mucosal epithelium. In the hope of obtaining such information, the number of larvae emerging from the intestines of preinfected and normal mice killed 36, 48, or 72 hr after receiving an oral challenge of third-stage larvae was determined. The results of three experiments are presented in Table III. It can be seen that significantly fewer larvae emerged from the intestines of immunized mice than from those of control mice. Moreover, there seemed to be some correlation between the degree of immunity expressed by the mice and the number of emergent larvae recovered. In Expt 1, for example, mice immunized with two or three minor infections (20 larvae per dose) showed a small reduction (19 and 29%, respectively) in the number of Day 5 cysts relative to the control value, and a slightly larger reduction (47 and 62%, respectively) in the number of
73
RECOVERY FROM MICE
Number of larvae recovered (mean ? SE) 87.6 k 12.3 5 9.8 k 100.7 +
20.3 1.4 1.1 16.3
” Twenty mice were each infected with 600 thirdstage larvae and killed after 48 hr. Half of the intestines were subjected to three consecutive 40-min incubations; they were transferred to new Petri dishes for each incubation. The remaining 10 intestines were given a single incubation (180 min).
48-hr p.c. emergent larvae. Immunization with a large number of third-stage larvae (three doses of 200 larvae; Chaicumpa et al. (1977); Prowse et al. (1978)) resulted in a greater degree of immunity (Expt 2, 78%) and a very marked reduction (>90%) in the number of larvae emerging from the isolated intestines (Expts 2 and 3). Viability
of Emergent Larvae
Providing the intestines were not incubated for an excessively long period (>60-90 min), the larvae recovered in the medium were intact and motile. To test their viability, larvae collected from the intestines of mice infected 48 hr previously were incubated at 37 C for several days in Medium 199 containing 10% fetal calf serum and 200 IU/ml of both penicillin and streptomycin sulfate (200 larvae in 15 ml of medium; loo-ml bottles, flat face down). During this period, the parasites continued to grow and differentiate. At least one molt was observed, the worms appeared to develop sexually and most were still alive after 6 days incubation (results not shown). The infective potential of emergent larvae was investigated by collecting larvae from the intestines of mice infected for different periods of time and administering them by stomach tube to normal mice. When the adoptive hosts were killed 10
74
EY, PROWSE,
AND JENKIN
TABLE III of Emergent Larvae of Hrligmosomoidrs po/q’g.y:.,‘rusfrom the Intestines of Normal and Preinfected Mice after an Oral Challenge with Third-Stage Larvae
Recovery
Experiment I”
Previous administrations of L:,
Number of emergent larvae” (hr postchallenge) Number of cysts” (Day 5 postchallenge)
None 2 x 20 3 x 20
210 2 3.3 171 + IO 150 + 13
2’
None 3 x 200
200 +- 9.0 43.2 k 7.4
3”
None 3 x 200
36 109 k I3 2.3 t 2.9
48 108 i 25 57 t 10 41 + IO 99 k 21 4.9 2 I.1 109 -t I6 3.4 -c I.5
72 119 k 15 15.1 t 4.6 -
” Mean ? SE (10 mice per group). Emergent larvae were collected over a 2-hr incubation. b Immunized mice were injected intravenously (iv) with 20 third-stage larvae (L:,) on Days -37 (3x infected only), -30, and - 16. All mice, including the controls. were purged with 3 mg of Pyrantel pamoate (Pfizer) on Days -8 and -6. Each was orally challenged on Day 0 with either 200 L:, (for cyst counts) or 600 L:, (for larval recovery). ’ Immunized mice received 200 L:, iv on Days -88, -56. and -40. All were given Pyrantel on Days -10 and -7 and challenged on Day 0 as in footnote b. ” Immunized mice received 200 L., iv on Days -53. -38, and -21. They were challenged on Day 0 as in footnote b.
days later, a proportion (1 l-36%) of the transferred parasites were found in the lumen of the small intestine as (apparently) mature adult worms (Table IV). The observed recoveries can be considered as minimal values of survival, since some of
TABLE IV Ability of Emergent Larvae of H~ligmosomoidr.~ polygyrus Collected at Different Times after Infection to Continue Development upon Oral Transfer to a Second Host Time after infection at which larvae were collected for transfer” (hr) 48 h 72 h 96 h 120 h 144 h Adult worms
Number’, of parasites present in the second host Day 3 (cysts) 15.2 + 3.5
Day IO (adults)
-
17.4 53.9 51.2 37.3 51.5
-
I I.5 f 3.9
’ I50 larvae or adult worms per mouse. Ir Mean I SE (five mice per group).
k + k 2 +
3.6 6.8 12.7 7.7 25.7
the transferred larvae may have been damaged during ejection through the syringe needle and passage through the stomach. The possibility of injury is exemplified by the very low survival of adult worms (used as a control) following similar adoptive transfer. These worms are adapted for existence within the lumen of the intestine, but they are much larger and they became entangled and blocked the needle during transfer. As a result, many must have been injured. DISCUSSION
The
cycle of Heligmosomoides has been studied in various detail by a number of investigators (Spurlock 1943; Baker 1954; Ehrenford 1954; Fahmy 1956; Dobson 1960), most recently by Bryant (1973) who has comprehensively reviewed the earlier data. After ingestion by the mouse, infective third-stage larvae exsheath in the stomach (Sommerville and Bailey 1973) and small intestine (Bryant 1973) before penetrating the mucosal epi-
polygyrus
life
Heligmosomoides
polygyrus:
thelium of the small intestine. By 24 hr, no larvae can be found in the lumen (Bryant 1973; Ey, unpublished data), indicating that most of them have penetrated the mucosa by this time. The larvae molt at approximately 90 hr p.i. (third to fourth stage) and 150 hr (fourth to fifth adult stage) and most of the worms return to the intestinal lumen by 190 hr to adopt their usual adult position (Bryant 1973). One of the limiting factors in investigations on the early parasitic stages of H. polygyrus has been the difficulty in excising individual larvae from the wall of the intestines of infected mice in numbers sufficient for study (e.g., Bryant 1973, 1974; Dennis 1976). This is a particular problem when one requires hundreds, if not thousands, of larvae for collecting secretions or extracts for immunochemical work. Attempts to culture the parasite in vitro have met with only partial success (Jones and Weinstein 1957; Sommerville and Weinstein 1964; Yasuraoka and Weinstein 1969). The results presented in this paper demonstrate that large numbers of larvae in the early parasitic stages of the life cycle can be recovered from infected mice with little effort. With a small amount of care, they can be collected virtually free from contamination with intestinal matter. The period for maximal recovery of larvae is between 40 and 100 hr p.i., although reasonable yields may be obtained at any time up to 6 or 7 days p.i. when the parasites begin to migrate back to the lumen. Most if not all of the emergent larvae collected at different times after infection were viable and many were able to continue their development under suitable conditions both in vitro and in vivo. At least some of the surviving parasites must penetrate the intestinal mucosa of the adoptive host in order to continue development, since encysted larvae were observed in the intestines of mice given 48-hr p.i. emergent larvae 3 days previously (Table IV). Whether or not the more developed larvae (e.g.,
RECOVERY FROM MICE
75
those collected 96-120 hr p.i.) must also penetrate the intestinal wall of an adoptive host to continue their development remains to be determined. The number of emergent larvae recovered from isolated intestines constituted a constant proportion of the dose of larvae given to the mice over a wide dose range (100-2400). Mice infected with up to 10,000 third-stage larvae have yielded a similar proportion of emergent larvae, although these mice do not survive more than a few days. Most of the larvae emerge from the intestines during the initial 40-60 min of incubation at 37 C. The phenomenon does not occur within the mouse but is apparently induced upon the death of the animal and isolation of the intestine. It is vital to commence the incubation as quickly as possible after killing the mouse and to restrict the incubation period to less than 1 hr if maximal recovery and viability is desired. Peristalsis usually continues for some time during the incubation and it is necessary to ligate both ends of the intestine to prevent contamination of the medium with digested matter. The results using immunized mice indicate that infective larvae must be arrested or immobilized soon after penetrating the mucosal lining of the small intestine. This was shown by the reduced number of larvae which emerged from the intestines of immunized compared with normal mice killed as early as 36 hr after challenge. Previous work from this laboratory has shown that the ability of infective larvae to penetrate the intestinal wall is unimpaired in immune mice (Chaicumpa et al. 1977). The reduction seems to bear some relationship to the degree of resistance expressed by the mice, i.e., to the proportion of larvae in a challenge dose which are killed by the mouse. A considerable period of time (l-3 weeks) is apparently required for the larvae to be killed, however, since immune mice treated with cortisone (an immunosuppressant) as late as lo-15 days after challenge exhibit
76
EY, PROWSE,
significantly increased worm burdens (Behnke and Parish 1979). The mechanisms responsible in immune mice for the developmental arrest, immobilization, and subsequent death of the Heligmasomoides pofygyrus larvae remain unclear. ACKNOWLEDGMENTS We are grateful to Mr. G. Ekserdjian and Ms. Ann Hallett for invaluable technical assistance, and to Dr. R. 1. Sommerville for allowing us to use his microscopes, for his interest in this work, and for helpful discussions. This work was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES
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