EXPERIMENTAL
42,
PARASITOLOGY
3443
( 1977)
Heligmosomoides polygyrus: Temporal, Population Characteristics
Spatial, and Morphological in LAF,/J Mice
RAYMOND H. CYPESS, HELEN L. LUCIA, JOHN L. ZIDIAN, AND CARMEN INES RIVERA-ORTIZ Departments
of Epidemiology and Microbiology, of Pittsburgh, Pittsburgh,
University
(Accepted
for publication
Graduate School of Public Health, Pennsylvania 22 September
15261,
U.S.A.
1976)
CYPESS, R. H., LUCIA, H. L., ZIDIAN, J. L., AND RIVERA-ORTIZ, C. I. 1977. Heligmosomo:des polygyrus: Temporal, spatial, and morphological population characteristics in LAF1/J mice. Experimental Parasitology 42, 3443. The course of an initial orally induced infection of Heligmosomoides polygyrus in the LAF, strain of mice was compared to that observed in A/He mice. In LAFl mice, the tissue-encysted larval-stage nematode lasts 1 to 2 days longer than in A/He mice. After larval emergence, the worms are not as successful in their anterior migration in LAF, mice as they are in A/He mice. After Day 14, adult worms are lost from LAF, mice, but not from A/He mice. Histologic study of the intestine did not reveal any significant difference between the mice, except to reflect the delay in larval emergence. The peripheral blood of the LAF, mice showed a leukocytosis on Days 4 and 6, while the A/He mice had a leukopenia from Days 4 through 20. There was no change in the differential count. No anatomic explanation for post developmental adult expulsion was found. I INDEX DESCRIPTORS: Heligmosomoides pozygyrus; Parasitic nematode; A/He; LAF1; Mice; Resistance; Histol’ogy; Leucocyte counts; Postdevelopmental adult expulsion.
INTRODUCTION
CBA,l and F1 of CsH X AKR ‘) require three or more infections before any form of resistance is observed (Zidian 1975). We have selected the LAFl strain for more detailed study because from Day 14 to Day 21 after an initial infection newly established adult stages are expelled. When these animals are rechallenged, adult stages fail to establish themselves in the intestine. In contrast, both parental strains of this hybrid (A/He and C57L) require three or more oral infections before a reduction in the number of adults is observed. The purpose of this paper, therefore, was to examine several temporal, spatial, and morphological characteristics of the parasite population and host responses in LAFl and A/He mice in order to determine pos-
Following
oral ingestion of HeUgmosomoides polygyrus larvae, several strains of mice develop resistance to reinfection to this gastrointestinal nematode. This resistance may be expressed at one or more phases of the life cycle of the parasite and the degree and type of resistance observed is influenced by the genetic composition of the host (Cypess and Zidian 1975). Thus, some inbred strains require only a single oral infection (SJL,l LAF1,l BLCF, I), some (Al, A/He,l BALB/C2 DBAz,l C57L,l C57BL/6,l B6D2F1 ‘) require two infections, while others (CsH,l AKR,l 1 Jackson Laboratory, Bar Harbor, Maine. 2 Charles River Breeding Laboratories, Wilmington, Massachusetts. 34 Cnoyrieht All rights
0
1977
by Academic
of reproduction
Press,
Inc.
in any form reserved.
ISSN
0014~-4894
~&@ZcX0&Lh
~OtfjgZJ?US: POPVLATION
CHARACTERISTICS
35
IN MICE
sible mechanisms responsible for the differences in expression of resistance between these two strains. MATERIALSANDMETHODS
The strain of Heligmosomoides polygyros, preparation of cultures, method of oral infection, and counting of adult worms have been previously described (Van Zandt 1961; Cypess and Zidian 1975). Female LAFl and A/He mice, 8 weeks of age, were given a single infection with 100 Heligmosomoides polygyrus larvae. The procedure for examining the intestines for worm distribution followed, with certain modifications, the method of Bawden (1969). At 4, 6, 8, 10, 12, 14, 16, 18, and 20 days after challenge, four mice from each strain were bled from the retro-orbital plexus, and then killed by cervical dislocation. Immediately after death, the intestines were removed, opened, and examined for the presence and distribution of larvae or adult stages. An equal number of mice from both groups were also immediateIy processed for histopathoIogica1 examination. In this latter group, the first 15 cm of the intestine were processed, 5 cm were fixed in 10% aqueous formalin, and 5 cm were fixed in 10% alcoholic formalin. Onethird of the animals had from 0 to 10 cm of the small intestine examined; another one-third had from 5-15 cm examined; and the remaining one-third had O-5 and lo15 cm examined. The fixed intestine was processed for routine hematoxylin and eosin staining and sectioned every 2.5 mm. Sections were 5-pm thick. Leukocyte Counts Total leukocyte counts were caIcuIated using the Unopette DisposabIe Pipetting System ( Becton-Dickinson and Co. ) . In this technique, cells were diluted to a final concentration of 1: 100 using a 3% glacial acetic acid solution as diluent. Differential leukocyte counts were calculated from thin blood smears after staining with Wright’s stain.
DAY FIG. 1. Mean number of tissue-stage gyrus present in the small intestine after fection of 100 larvae.
H. poltjone in-
RESULTS
At 6 days, a11 of the Heligmosomoides polygyrus observed in both mouse strains were larvae-encysted in the muscularis of the small intestine (Fig. 1). Equal numbers of larvae were observed in both strains (68; range 59-84). At 8 days, however, the mean number of larvae (71.6; range 6378) in the LAFl was significantly higher than the number observed in the A/He (23.6; range 14-30) (Fig. 1). At this time, however, no adult worms were observed in the intestinal lumen of the LAFr, although 31.8 (range 1347) of the parasites had emerged in the A/He (Fig. 2). At 10 days, a11the worms (66; range 53-75) detected in the A/He were adults, but a small number (5; range O-10) of encysted larvae were still present in the LAFl (Figs. 1 and 2). At Days 12 and 14 the mean number of adults present in the A/He (64.2 and 64.4) and the LAF, (60.2 and 68.6) were similar, but between 16 and 20 days a gradual loss in the number of adults was observed in the LAFr but not in the A/He (Fig. 2). Between Days 12 and 20, equal numbers of male and female worms were observed in both the A/He and LAFl strains (Table 1). In both strains the majority of encysted larvae were detected in the first 10 cm of the small intestine (Fig. 3). After larval
CYPESS ET AL.
36
i
lb
ih
lb
20
DAY
FIG. 2. Mean number of H. polygyrus present in the small intestine after one infection with 100 larvae.
emergence, however, the distribution of adult stages in the first 40 cm of the small intestine varied significantly between the two strains (Fig. 4). Thus in the A/He during this l2-day time span, 89.5% of the total number of adults were found in the first 5 cm, 7.9% in the second 5-cm segment, 2.1% in the third 5cm segment, and 0.7% in the fourth 5cm segment. In contrast, in the LAF1, 49.2% of the total number of the adults were found in the first 5 cm, 20.9% in the second, 16.3% in the third, and 14.7% in the 4th.
through the muscularis around the cyst (Fig. 5). By Day 6 the larvae in the A/He mice were larger in cross section than on Day 4. A second internal tubular structure, the protogonad, had appeared and a prominent cuticle with striations was seen. The cysts contained clear fluid, a few scattered PMNS and macrophages, and the lining and wall of the cyst remained similar to that seen on Day 4. The cysts had enlarged and frequently extensive destruction of both the muscularis interna and externa was noted. In the LAP1 on Day 6, one-third of the cysts resembled those seen in the A/HeJ mice, but two-thirds of the cysts contained, besides the normal healthy maturing larva, a large quantity of PMNS. The wall of the cyst had become moderately infiltrated with PMNS, and the cyst lining was difficult to define (Fig. 6). By Day 8, most of the larvae had matured, left the cysts, and were present
Histologic Description and Comparison of the LAFl and A/He Strailzs On Day 4, no difference in the histology of the LAFl and A/He animals was seen. Larvae were coiled within cysts, located within the muscularis of the mouse intestine. In cross section, the larva contained a central circular gut lumen, a cle.ar coelom, and a muscular body wall, with an easily visible cuticle on its surface. The cyst contained a larva and a quantity of clear fluid, within which were floating a few leukocyte scattered polymorphonuclear (PMNS ) and macrophages. The lining of the cyst was a single cell layer of fibrocytes, and the wall of the cyst consisted of smooth muscle of the muscularis interna and externa. A very mild inflammatory infiltrate, varying from l-2 PMNS, to 20-30 PMNS and macrophages, was scattered
cm
100 I
% 50
DAY IO
,lJlJ-L+ 5
IO
15
20
cm
q
A/HeJ
0
LAF,
FIG. 3. Percent distribution larvae in the small intestine.
of N. pdygyrus
Heligmosomoides
polygyrus:
POPULATION
within the intestinal lumen in the A/He. The empty cysts were abscessed and contained densely packed PMNS and macrophages. Only a few eosinophils were noted (Fig. 7). The adult worms in the intestinal lumen appeared undamaged and no host reaction was present within the intestinal tissues. A few of the stages, however, were still present as larvae within an uninflamed cyst, similar to that seen on Day 6. Tn the LAF1, in contrast, on Day 8 only 50% of the larvae had emerged from their cysts. The remaining 50% ‘of the worms were larvae still contained within cysts and were similar in appearance to those seen on Day 6. In the A/He by Day 10, the abscesses within the intestinal wall had started to organize, with fibroblasts and macrophages invading the periphery. A sprinkle of eosinophils, and a giant cell or two had appeared. 100,
100,
DAY 6
% 5 0 L-
%
DAY 8
50
0
0 5
10
15
20
cm 100
%
100
5 0
%
50
0
0
100
100
% 50
% 50
0
0
100
100
% 50
% 50
0
0 5
io
15 20 cm
5
IO
15 20 cm
37
CHARACTERISTLCS IN MICE TABLE
I
Number of Male and Female Adult Neligmosomoides polygyrus Nematodes Day
Strain
12
A/He LAFl A/He LAFl A/He LAF, A/He LAFl A/He LAFl
14 16 18 20
Male
34.8 28.8 32.2 34.4 33.G 24.2 33.2 19.8 29.4 13.0
Female
Male/ Female
29.6 31.4 32.8 34.8 39.8 26.2 33.0 18.8 36.0 13.0
1.17 0.92 1.00 0.99 0.83 0.92 1.01 1.05 0.82 1.00
In the LAF1, by Day 10, a majority of the cysts had become empty abscesses, with peripheral organization, similar to those seen in A/He animals. However, a small number of the cysts still contained larvae, and were similar to those seen in the LAFr animals on Day 8. By Day 12, no difference could be seen between LAFl and A/He animals. The intestinal wall lesions were organizing abscesses similar to those seen on Day 10, except that a few lymphocytes sometimes appeared at the periphery of the lesion. Between Days 14 and 20, the abscesses in the intestinal walls showed progressive organization. The LAFr animaIs seemed to resorb the necrotic material from the center of the abscess earher than the A/He, replacing the destroyed muscle with a scar of fibrous tissue and a few scattered inflammatory cells. The abscesses in the A/ He mice remained sizabIe through Days 16 to 20. From Days 8 to 20 adults couId be found within the mouse intestina1 lumen in both strains. They appeared undamaged, and no host reaction could be seen (Fig. 8). This was true even in the LAF, at the time when the adu‘lts were being expelled. Peripheral Blood Changes
FIG. 4. Percent distribution gyros in the small intestine.
of adult H.
poly-
In the LAF1, a marked peripheral leukocytosis was noted on Days 4 and 6, but
38
CYPESS
FIG.
5. Heligmosomoides
ET
AL.
polygyrus larva, Day 4, encysted within
small intestine muscularis.
X78.75.
the total leukocyte count returned to preinft :ction levels on Days 8 through 20. In COPktrast, a marked leukopenia beginning
FIG.
X78.75.
6. Heligmosomoides
on Day 4 and extending through Day 20 was observed in the A/He (Fig. 9). In both strains no significant difference in the
polygyrus larva, Day 6, showing
increased inflammatory
infiltrate.
Heligmosomoides
polygyrus:
POPULATION
CHARACTERISTICS
IN
MICE
F'IG. 7. Day 8, abscess located within the small intestinal muscularis, marking former site of He1 igmosomoides polygyrus larval encystment. X78.75.
FIG. 8. Day 8, adult Heligmosomoides polygyrus, within small intestinal is no obvious inflammatory response in the nearby villi. ~78.75.
lumen. Note there
34
40
CXPESS
ET
AL.
ment, these cysts are filled with a clear fluid, the source and nature of which are unknown. 13.0 In the LAF1, as early as Day 6, the cysts contained a greater number of inflammatory cells and both the tissue and peripheral blood response to the developing parab :: 9.0 site was greater in this strain. In previous 2 8.0 studies, using Swiss-Webster mice, resisA tance was associated with an accelerated -I 7.0 and prolonged peripheral leukocytosis (Cy2c 6.0 pess 1972). However, in the LAFl the Ieu5.0 kocytosis observed between Days 4 and 6 4.0 had subsided by the time of preadult emer0 2 4 6 8 IO 12 14 16 18 20 gence and adult expulsion. DAYS Two additional expressions of resistance FIG. 9. Total leukocyte counts observed in LAFI were observed in the LAF1; the gradual and A/He mice following infection with 100 H. loss of adult stages between Days 14 and polygyrus larvae. 20 (postdevelopmental adult expulsion), and the failure of preadult stages to mipercentage of lymphocytes, neutrophils, grate anteriorly in the_ small intestine folmonocytes, or eosinophils was detected throughout the experimental period. lowing emergence. Postdevelopmental adult expulsion 3 is a consistent expression of resistance observed in rodents infected with DISCUSSION either Trichinellu spiruZis (Larsh and Race Ingestion of third-stage Heligmosomoides poZygyrus larvae results in the ex- 1954), Nippostrongylus brasiliensis (Haley 1962), Trichostrongylus colubriformis posure of susceptible hosts to a variety ( Herlich, Douvres, and Isenstein 1956)) of somatic and metabolic antigens assoand Trichuris muris (Wakelin 1967), but ciated with the parasitic life cycle stages. is seldom observed following infection with The type and degree of host response to Heligmosomoides polygyrus in a variety each of these stages and their antigens will be influenced by the location of these of mouse strains (Zidian 1975). In most stages within the host, the duration of ex- mouse strains following adult emergence, posure to each of these stages, the immuno- the majority of Heligmosomoides polygyrus genicity of the stages themselves, and the adults are found just distal to the pyloric history of previous exposure to the agent. sphincter. In contrast, in the LAFl between Days 14 and 22, there was a gradual With these factors as points of reference, the life cycle of Heligmosomoides polyloss of adults from all segments of the gyros can be divided into six phases (Fig. gut and the adult population present was 10). distributed more uniformly throughout the After an initial infection, resistance in first 40 cm, suggesting that many of the the LAFl strain is noted first as a delay adults were unable to successfully establish in parasite emergence by the preadult from themselves. Resistance in this case would the muscularis. This effect appeared to be 3 Postdevelopmental adult expulsion has been related to life cycle events associated with However, classic self-cure refers the development of larvae within the cyst termed, self-cure. to the loss of a residual population of adults foland/or the subsequent release of preadult lowing exposure to developmental stages (St011 stages from cysts into the intestinal lumen 1929; Cypess and Van Zandt 1973; Ogilvie and (phases III-VI). During larval develop- Jones 1973). 14.0
LAF,
L-4
A/He
u
Heligmosomoides
poZygyrus:
POPULATION
CHARACTERISTICS
IN
MICE
41
42
CYPESS ET AL.
appear to be related to factors that prevent the anterior migration and establishment of the newly emerged stage. In most studies an unbalanced sex ratio in favor of females has been observed in a variety of nematode infections (Lin and Olson 1972). In the case of NippostrongyZZJUS where there is also a pronounced proximal shift in the distribution of adult stages in the small intestine, female worms appear to have a positive effect upon the anteriad migration of male worms (Brambell 1965; Gimenez and Roche 1972). In our studies, between Days 10 and 20, no significant difference in sex ratio of Heligmosomoides polygyrus was noted between resistant and control mice, suggesting that the failure of the adult worms to migrate proximally in LAFl mice was not related to alterations in the adult sex ratios. Since we could not distinguish gross morphological differences between the adult populations in either mouse strain, it would appear that if changes in the parasite are responsible for the inability of the adults to migrate and establish in the LAF1, these changes are subtle and may involve alterations in the metabolic or secretory pathways of the parasites. The postdevelopmental adult expulsion observed in infections with Trichinella spiralis, Nippostrongyluq bras&~&, and Trichostrongylus colubriformis is associated with an intense allergic inflammatory response in the small intestine (Larsh and Race 1954; Taliaferro and Sarles 1939; Wells 1962; Jarrett, Jarrett, Miller, and Urquhart 1968; Rothwell and Dineen 1972). In our model there was little evidence of tissue reaction associated with the adult stages and no difference in intestinal histology surrounding the adults was noted between the two strains. The tissue reactions around the developing cysts were of the nonspecific type with few eosinophils and mast cells evident, which prevents us from assigning it with cetiainty to any one type of immunologic response. Therefore, the mechanisms responsible for postdevelopmental adult
expulsion to Heligmosomoides may be different.
polygyrus
ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. Meryl Karol for her advice in the course of this work, and to Ms. Doris LaPietra and Mrs. Rebecca Dombroske for their technical assistance. This research has been aided by NIH Grant AI 10490; Dr. Rivera is recipient of special NIH postdoctoral fellowship lF22 AI 00755 01; Dr. Cypess is a recipient of Career Development Award lK04 AI 00056 02.
REFERENCES BAWDEN, R. J, 1969. Some effects of the diet of mice on New~~tospiroides dubizrs (Nematoda) . Parasitology 59, 203-213. BRAMBELL, M. R. 19’65. The distribution of a primary infestation of Nippostrongylus brasiliensis in the small intestine of laboratory rats. Parasitology 55, 313-324. CYPESS, R. H. 1972. Bl’ood cell count changes associated with immunity to Nematospiroides dubius. Journal of Parasitology 58, 563566. CYPESS, R. H., AND VAN ZANDT, P. 1973. Time studies on the immune expulsion of Nematospiroides dubius in the mouse. Journal of Parasitology 59, 579-580. CYPESS, R. H., AND ZIDIAN, J, L. 1975. Heligmosomodes polygyrus (Nemafiospiroides dubius) : The development of self-cure and/or protection in several strains of mice. journal of Parasitology 61,819-824. GIMENEZ, A., AND ROCHE, M. 1972. Influence of male and female Nippostrongylus bras&r&s on each other’s distribution in the intestine of the rat. Parasitology 64,305-310. HALEY, A. J. 1962. Biol’ogy of the rat nematode, Nippostrongylus brasiliensis (Travassos, 1914). II. Preparasitic stages and development in the laboratory rat. journal of Parasitology 48, 13-23. HERLICH, H., DOUVRES, F. W., AND ISENSTEIN, R. S. 1956. Experimental infections of guinea pigs with Trichostrongylus colubriformis, a parasite of ruminants. Proceedings of the Helminthological Society of Washington 23, 104-105. JARRETT, W. F. H., JARRETT, E. E., MILLER, H. R. P., AND URQUHART, G. M. 1968. Quantitative studies on the mechanism of self-cure in Nippostrongylus brasiliensis infections. In “The Reaction of the Host to Parasitism” (E. J. L. Soulsby, ed. ), Academic Press, New York. LARSH, J. E., JR., AND RACE, G. J. 1954. A histopathologic study of the anterior small intestine of immunized and non-immunized mice infected with Trichinella spiralis. Journal of Infectious Diseases 94, 262-272.
&?~i@7ZOW7’LOid~S
jdZJYgy?US:
POPULATION
LIN, T.-M., AND OLSON, L. J. 1972. Sex ratio of Trichinella spiralis in guinea pigs and mice in relation to host resistance. Proceedings of the Helminthological Society of Washington 39, 169-172. OGILVIE, B. M., AND JONES, v. E. 1973. hmUUity in the parasitic relationship between helminths and hosts. Progress in Allergy 17, 93-144. ROTHWELL, T. L. W., AND DINEEN, J. K. 1972. Cellular reactions in guinea pigs following primary and challenged infection with Trichostrongylus colubriformis with special reference to the roles played by eosinophils and basophils in rejection of the parasite. Immunology 22, 733-745. STOLL, N. R. 1929. Studies with the strongylid nematode, Haemonchus contortus. I. Acquired resistance ‘of hosts under natural reinfection conditions out-of-doors. American Journal of Hygiene 10,384-4X
CHARACTERISTICS
IN
MICE
43
TALKFERRO, W. H., AND SARLES, M. P.1939. The cellular reactions in the skin, lungs and intestine of normal and immune rats after infection with Nippostrongylus muris. .lournul of Infectious Diseases 64, 157-162. VAN ZANDT, P. D. 1961. Studies on the immunity relationships in white mice given infections with Nematospiroides dubius Baylis, 1926 (Nematoda: Heligmosomidae) . Journal of the Elisha Mitchell Science So&@ 77, 300-309. WAKELIN, D. 1967. Acquired immunity to Trichuris mu& in the albino laboratory mouse. Parasitology 57, 515-524. WELLS, I’. D. 1962. Mast cell, eosinophil and histamine levels in Nippostrongylus brasiliensis infected rats. Experimental Parasitology 12, 82. ZIDIAN, J. L. 1975. The influence of host genetic strain on resistance to Heligmosomoides polygyrus in the mouse. Masters thesis. University of Pittsburgh.
42,
PARASITOLOGY
3443
( 1977)
Heligmosomoides polygyrus: Temporal, Population Characteristics
Spatial, and Morphological in LAF,/J Mice
RAYMOND H. CYPESS, HELEN L. LUCIA, JOHN L. ZIDIAN, AND CARMEN INES RIVERA-ORTIZ Departments
of Epidemiology and Microbiology, of Pittsburgh, Pittsburgh,
University
(Accepted
for publication
Graduate School of Public Health, Pennsylvania 22 September
15261,
U.S.A.
1976)
CYPESS, R. H., LUCIA, H. L., ZIDIAN, J. L., AND RIVERA-ORTIZ, C. I. 1977. Heligmosomo:des polygyrus: Temporal, spatial, and morphological population characteristics in LAF1/J mice. Experimental Parasitology 42, 3443. The course of an initial orally induced infection of Heligmosomoides polygyrus in the LAF, strain of mice was compared to that observed in A/He mice. In LAFl mice, the tissue-encysted larval-stage nematode lasts 1 to 2 days longer than in A/He mice. After larval emergence, the worms are not as successful in their anterior migration in LAF, mice as they are in A/He mice. After Day 14, adult worms are lost from LAF, mice, but not from A/He mice. Histologic study of the intestine did not reveal any significant difference between the mice, except to reflect the delay in larval emergence. The peripheral blood of the LAF, mice showed a leukocytosis on Days 4 and 6, while the A/He mice had a leukopenia from Days 4 through 20. There was no change in the differential count. No anatomic explanation for post developmental adult expulsion was found. I INDEX DESCRIPTORS: Heligmosomoides pozygyrus; Parasitic nematode; A/He; LAF1; Mice; Resistance; Histol’ogy; Leucocyte counts; Postdevelopmental adult expulsion.
INTRODUCTION
CBA,l and F1 of CsH X AKR ‘) require three or more infections before any form of resistance is observed (Zidian 1975). We have selected the LAFl strain for more detailed study because from Day 14 to Day 21 after an initial infection newly established adult stages are expelled. When these animals are rechallenged, adult stages fail to establish themselves in the intestine. In contrast, both parental strains of this hybrid (A/He and C57L) require three or more oral infections before a reduction in the number of adults is observed. The purpose of this paper, therefore, was to examine several temporal, spatial, and morphological characteristics of the parasite population and host responses in LAFl and A/He mice in order to determine pos-
Following
oral ingestion of HeUgmosomoides polygyrus larvae, several strains of mice develop resistance to reinfection to this gastrointestinal nematode. This resistance may be expressed at one or more phases of the life cycle of the parasite and the degree and type of resistance observed is influenced by the genetic composition of the host (Cypess and Zidian 1975). Thus, some inbred strains require only a single oral infection (SJL,l LAF1,l BLCF, I), some (Al, A/He,l BALB/C2 DBAz,l C57L,l C57BL/6,l B6D2F1 ‘) require two infections, while others (CsH,l AKR,l 1 Jackson Laboratory, Bar Harbor, Maine. 2 Charles River Breeding Laboratories, Wilmington, Massachusetts. 34 Cnoyrieht All rights
0
1977
by Academic
of reproduction
Press,
Inc.
in any form reserved.
ISSN
0014~-4894
~&@ZcX0&Lh
~OtfjgZJ?US: POPVLATION
CHARACTERISTICS
35
IN MICE
sible mechanisms responsible for the differences in expression of resistance between these two strains. MATERIALSANDMETHODS
The strain of Heligmosomoides polygyros, preparation of cultures, method of oral infection, and counting of adult worms have been previously described (Van Zandt 1961; Cypess and Zidian 1975). Female LAFl and A/He mice, 8 weeks of age, were given a single infection with 100 Heligmosomoides polygyrus larvae. The procedure for examining the intestines for worm distribution followed, with certain modifications, the method of Bawden (1969). At 4, 6, 8, 10, 12, 14, 16, 18, and 20 days after challenge, four mice from each strain were bled from the retro-orbital plexus, and then killed by cervical dislocation. Immediately after death, the intestines were removed, opened, and examined for the presence and distribution of larvae or adult stages. An equal number of mice from both groups were also immediateIy processed for histopathoIogica1 examination. In this latter group, the first 15 cm of the intestine were processed, 5 cm were fixed in 10% aqueous formalin, and 5 cm were fixed in 10% alcoholic formalin. Onethird of the animals had from 0 to 10 cm of the small intestine examined; another one-third had from 5-15 cm examined; and the remaining one-third had O-5 and lo15 cm examined. The fixed intestine was processed for routine hematoxylin and eosin staining and sectioned every 2.5 mm. Sections were 5-pm thick. Leukocyte Counts Total leukocyte counts were caIcuIated using the Unopette DisposabIe Pipetting System ( Becton-Dickinson and Co. ) . In this technique, cells were diluted to a final concentration of 1: 100 using a 3% glacial acetic acid solution as diluent. Differential leukocyte counts were calculated from thin blood smears after staining with Wright’s stain.
DAY FIG. 1. Mean number of tissue-stage gyrus present in the small intestine after fection of 100 larvae.
H. poltjone in-
RESULTS
At 6 days, a11 of the Heligmosomoides polygyrus observed in both mouse strains were larvae-encysted in the muscularis of the small intestine (Fig. 1). Equal numbers of larvae were observed in both strains (68; range 59-84). At 8 days, however, the mean number of larvae (71.6; range 6378) in the LAFl was significantly higher than the number observed in the A/He (23.6; range 14-30) (Fig. 1). At this time, however, no adult worms were observed in the intestinal lumen of the LAFr, although 31.8 (range 1347) of the parasites had emerged in the A/He (Fig. 2). At 10 days, a11the worms (66; range 53-75) detected in the A/He were adults, but a small number (5; range O-10) of encysted larvae were still present in the LAFl (Figs. 1 and 2). At Days 12 and 14 the mean number of adults present in the A/He (64.2 and 64.4) and the LAF, (60.2 and 68.6) were similar, but between 16 and 20 days a gradual loss in the number of adults was observed in the LAFr but not in the A/He (Fig. 2). Between Days 12 and 20, equal numbers of male and female worms were observed in both the A/He and LAFl strains (Table 1). In both strains the majority of encysted larvae were detected in the first 10 cm of the small intestine (Fig. 3). After larval
CYPESS ET AL.
36
i
lb
ih
lb
20
DAY
FIG. 2. Mean number of H. polygyrus present in the small intestine after one infection with 100 larvae.
emergence, however, the distribution of adult stages in the first 40 cm of the small intestine varied significantly between the two strains (Fig. 4). Thus in the A/He during this l2-day time span, 89.5% of the total number of adults were found in the first 5 cm, 7.9% in the second 5-cm segment, 2.1% in the third 5cm segment, and 0.7% in the fourth 5cm segment. In contrast, in the LAF1, 49.2% of the total number of the adults were found in the first 5 cm, 20.9% in the second, 16.3% in the third, and 14.7% in the 4th.
through the muscularis around the cyst (Fig. 5). By Day 6 the larvae in the A/He mice were larger in cross section than on Day 4. A second internal tubular structure, the protogonad, had appeared and a prominent cuticle with striations was seen. The cysts contained clear fluid, a few scattered PMNS and macrophages, and the lining and wall of the cyst remained similar to that seen on Day 4. The cysts had enlarged and frequently extensive destruction of both the muscularis interna and externa was noted. In the LAP1 on Day 6, one-third of the cysts resembled those seen in the A/HeJ mice, but two-thirds of the cysts contained, besides the normal healthy maturing larva, a large quantity of PMNS. The wall of the cyst had become moderately infiltrated with PMNS, and the cyst lining was difficult to define (Fig. 6). By Day 8, most of the larvae had matured, left the cysts, and were present
Histologic Description and Comparison of the LAFl and A/He Strailzs On Day 4, no difference in the histology of the LAFl and A/He animals was seen. Larvae were coiled within cysts, located within the muscularis of the mouse intestine. In cross section, the larva contained a central circular gut lumen, a cle.ar coelom, and a muscular body wall, with an easily visible cuticle on its surface. The cyst contained a larva and a quantity of clear fluid, within which were floating a few leukocyte scattered polymorphonuclear (PMNS ) and macrophages. The lining of the cyst was a single cell layer of fibrocytes, and the wall of the cyst consisted of smooth muscle of the muscularis interna and externa. A very mild inflammatory infiltrate, varying from l-2 PMNS, to 20-30 PMNS and macrophages, was scattered
cm
100 I
% 50
DAY IO
,lJlJ-L+ 5
IO
15
20
cm
q
A/HeJ
0
LAF,
FIG. 3. Percent distribution larvae in the small intestine.
of N. pdygyrus
Heligmosomoides
polygyrus:
POPULATION
within the intestinal lumen in the A/He. The empty cysts were abscessed and contained densely packed PMNS and macrophages. Only a few eosinophils were noted (Fig. 7). The adult worms in the intestinal lumen appeared undamaged and no host reaction was present within the intestinal tissues. A few of the stages, however, were still present as larvae within an uninflamed cyst, similar to that seen on Day 6. Tn the LAF1, in contrast, on Day 8 only 50% of the larvae had emerged from their cysts. The remaining 50% ‘of the worms were larvae still contained within cysts and were similar in appearance to those seen on Day 6. In the A/He by Day 10, the abscesses within the intestinal wall had started to organize, with fibroblasts and macrophages invading the periphery. A sprinkle of eosinophils, and a giant cell or two had appeared. 100,
100,
DAY 6
% 5 0 L-
%
DAY 8
50
0
0 5
10
15
20
cm 100
%
100
5 0
%
50
0
0
100
100
% 50
% 50
0
0
100
100
% 50
% 50
0
0 5
io
15 20 cm
5
IO
15 20 cm
37
CHARACTERISTLCS IN MICE TABLE
I
Number of Male and Female Adult Neligmosomoides polygyrus Nematodes Day
Strain
12
A/He LAFl A/He LAFl A/He LAF, A/He LAFl A/He LAFl
14 16 18 20
Male
34.8 28.8 32.2 34.4 33.G 24.2 33.2 19.8 29.4 13.0
Female
Male/ Female
29.6 31.4 32.8 34.8 39.8 26.2 33.0 18.8 36.0 13.0
1.17 0.92 1.00 0.99 0.83 0.92 1.01 1.05 0.82 1.00
In the LAF1, by Day 10, a majority of the cysts had become empty abscesses, with peripheral organization, similar to those seen in A/He animals. However, a small number of the cysts still contained larvae, and were similar to those seen in the LAFr animals on Day 8. By Day 12, no difference could be seen between LAFl and A/He animals. The intestinal wall lesions were organizing abscesses similar to those seen on Day 10, except that a few lymphocytes sometimes appeared at the periphery of the lesion. Between Days 14 and 20, the abscesses in the intestinal walls showed progressive organization. The LAFr animaIs seemed to resorb the necrotic material from the center of the abscess earher than the A/He, replacing the destroyed muscle with a scar of fibrous tissue and a few scattered inflammatory cells. The abscesses in the A/ He mice remained sizabIe through Days 16 to 20. From Days 8 to 20 adults couId be found within the mouse intestina1 lumen in both strains. They appeared undamaged, and no host reaction could be seen (Fig. 8). This was true even in the LAF, at the time when the adu‘lts were being expelled. Peripheral Blood Changes
FIG. 4. Percent distribution gyros in the small intestine.
of adult H.
poly-
In the LAF1, a marked peripheral leukocytosis was noted on Days 4 and 6, but
38
CYPESS
FIG.
5. Heligmosomoides
ET
AL.
polygyrus larva, Day 4, encysted within
small intestine muscularis.
X78.75.
the total leukocyte count returned to preinft :ction levels on Days 8 through 20. In COPktrast, a marked leukopenia beginning
FIG.
X78.75.
6. Heligmosomoides
on Day 4 and extending through Day 20 was observed in the A/He (Fig. 9). In both strains no significant difference in the
polygyrus larva, Day 6, showing
increased inflammatory
infiltrate.
Heligmosomoides
polygyrus:
POPULATION
CHARACTERISTICS
IN
MICE
F'IG. 7. Day 8, abscess located within the small intestinal muscularis, marking former site of He1 igmosomoides polygyrus larval encystment. X78.75.
FIG. 8. Day 8, adult Heligmosomoides polygyrus, within small intestinal is no obvious inflammatory response in the nearby villi. ~78.75.
lumen. Note there
34
40
CXPESS
ET
AL.
ment, these cysts are filled with a clear fluid, the source and nature of which are unknown. 13.0 In the LAF1, as early as Day 6, the cysts contained a greater number of inflammatory cells and both the tissue and peripheral blood response to the developing parab :: 9.0 site was greater in this strain. In previous 2 8.0 studies, using Swiss-Webster mice, resisA tance was associated with an accelerated -I 7.0 and prolonged peripheral leukocytosis (Cy2c 6.0 pess 1972). However, in the LAFl the Ieu5.0 kocytosis observed between Days 4 and 6 4.0 had subsided by the time of preadult emer0 2 4 6 8 IO 12 14 16 18 20 gence and adult expulsion. DAYS Two additional expressions of resistance FIG. 9. Total leukocyte counts observed in LAFI were observed in the LAF1; the gradual and A/He mice following infection with 100 H. loss of adult stages between Days 14 and polygyrus larvae. 20 (postdevelopmental adult expulsion), and the failure of preadult stages to mipercentage of lymphocytes, neutrophils, grate anteriorly in the_ small intestine folmonocytes, or eosinophils was detected throughout the experimental period. lowing emergence. Postdevelopmental adult expulsion 3 is a consistent expression of resistance observed in rodents infected with DISCUSSION either Trichinellu spiruZis (Larsh and Race Ingestion of third-stage Heligmosomoides poZygyrus larvae results in the ex- 1954), Nippostrongylus brasiliensis (Haley 1962), Trichostrongylus colubriformis posure of susceptible hosts to a variety ( Herlich, Douvres, and Isenstein 1956)) of somatic and metabolic antigens assoand Trichuris muris (Wakelin 1967), but ciated with the parasitic life cycle stages. is seldom observed following infection with The type and degree of host response to Heligmosomoides polygyrus in a variety each of these stages and their antigens will be influenced by the location of these of mouse strains (Zidian 1975). In most stages within the host, the duration of ex- mouse strains following adult emergence, posure to each of these stages, the immuno- the majority of Heligmosomoides polygyrus genicity of the stages themselves, and the adults are found just distal to the pyloric history of previous exposure to the agent. sphincter. In contrast, in the LAFl between Days 14 and 22, there was a gradual With these factors as points of reference, the life cycle of Heligmosomoides polyloss of adults from all segments of the gyros can be divided into six phases (Fig. gut and the adult population present was 10). distributed more uniformly throughout the After an initial infection, resistance in first 40 cm, suggesting that many of the the LAFl strain is noted first as a delay adults were unable to successfully establish in parasite emergence by the preadult from themselves. Resistance in this case would the muscularis. This effect appeared to be 3 Postdevelopmental adult expulsion has been related to life cycle events associated with However, classic self-cure refers the development of larvae within the cyst termed, self-cure. to the loss of a residual population of adults foland/or the subsequent release of preadult lowing exposure to developmental stages (St011 stages from cysts into the intestinal lumen 1929; Cypess and Van Zandt 1973; Ogilvie and (phases III-VI). During larval develop- Jones 1973). 14.0
LAF,
L-4
A/He
u
Heligmosomoides
poZygyrus:
POPULATION
CHARACTERISTICS
IN
MICE
41
42
CYPESS ET AL.
appear to be related to factors that prevent the anterior migration and establishment of the newly emerged stage. In most studies an unbalanced sex ratio in favor of females has been observed in a variety of nematode infections (Lin and Olson 1972). In the case of NippostrongyZZJUS where there is also a pronounced proximal shift in the distribution of adult stages in the small intestine, female worms appear to have a positive effect upon the anteriad migration of male worms (Brambell 1965; Gimenez and Roche 1972). In our studies, between Days 10 and 20, no significant difference in sex ratio of Heligmosomoides polygyrus was noted between resistant and control mice, suggesting that the failure of the adult worms to migrate proximally in LAFl mice was not related to alterations in the adult sex ratios. Since we could not distinguish gross morphological differences between the adult populations in either mouse strain, it would appear that if changes in the parasite are responsible for the inability of the adults to migrate and establish in the LAF1, these changes are subtle and may involve alterations in the metabolic or secretory pathways of the parasites. The postdevelopmental adult expulsion observed in infections with Trichinella spiralis, Nippostrongyluq bras&~&, and Trichostrongylus colubriformis is associated with an intense allergic inflammatory response in the small intestine (Larsh and Race 1954; Taliaferro and Sarles 1939; Wells 1962; Jarrett, Jarrett, Miller, and Urquhart 1968; Rothwell and Dineen 1972). In our model there was little evidence of tissue reaction associated with the adult stages and no difference in intestinal histology surrounding the adults was noted between the two strains. The tissue reactions around the developing cysts were of the nonspecific type with few eosinophils and mast cells evident, which prevents us from assigning it with cetiainty to any one type of immunologic response. Therefore, the mechanisms responsible for postdevelopmental adult
expulsion to Heligmosomoides may be different.
polygyrus
ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. Meryl Karol for her advice in the course of this work, and to Ms. Doris LaPietra and Mrs. Rebecca Dombroske for their technical assistance. This research has been aided by NIH Grant AI 10490; Dr. Rivera is recipient of special NIH postdoctoral fellowship lF22 AI 00755 01; Dr. Cypess is a recipient of Career Development Award lK04 AI 00056 02.
REFERENCES BAWDEN, R. J, 1969. Some effects of the diet of mice on New~~tospiroides dubizrs (Nematoda) . Parasitology 59, 203-213. BRAMBELL, M. R. 19’65. The distribution of a primary infestation of Nippostrongylus brasiliensis in the small intestine of laboratory rats. Parasitology 55, 313-324. CYPESS, R. H. 1972. Bl’ood cell count changes associated with immunity to Nematospiroides dubius. Journal of Parasitology 58, 563566. CYPESS, R. H., AND VAN ZANDT, P. 1973. Time studies on the immune expulsion of Nematospiroides dubius in the mouse. Journal of Parasitology 59, 579-580. CYPESS, R. H., AND ZIDIAN, J, L. 1975. Heligmosomodes polygyrus (Nemafiospiroides dubius) : The development of self-cure and/or protection in several strains of mice. journal of Parasitology 61,819-824. GIMENEZ, A., AND ROCHE, M. 1972. Influence of male and female Nippostrongylus bras&r&s on each other’s distribution in the intestine of the rat. Parasitology 64,305-310. HALEY, A. J. 1962. Biol’ogy of the rat nematode, Nippostrongylus brasiliensis (Travassos, 1914). II. Preparasitic stages and development in the laboratory rat. journal of Parasitology 48, 13-23. HERLICH, H., DOUVRES, F. W., AND ISENSTEIN, R. S. 1956. Experimental infections of guinea pigs with Trichostrongylus colubriformis, a parasite of ruminants. Proceedings of the Helminthological Society of Washington 23, 104-105. JARRETT, W. F. H., JARRETT, E. E., MILLER, H. R. P., AND URQUHART, G. M. 1968. Quantitative studies on the mechanism of self-cure in Nippostrongylus brasiliensis infections. In “The Reaction of the Host to Parasitism” (E. J. L. Soulsby, ed. ), Academic Press, New York. LARSH, J. E., JR., AND RACE, G. J. 1954. A histopathologic study of the anterior small intestine of immunized and non-immunized mice infected with Trichinella spiralis. Journal of Infectious Diseases 94, 262-272.
&?~i@7ZOW7’LOid~S
jdZJYgy?US:
POPULATION
LIN, T.-M., AND OLSON, L. J. 1972. Sex ratio of Trichinella spiralis in guinea pigs and mice in relation to host resistance. Proceedings of the Helminthological Society of Washington 39, 169-172. OGILVIE, B. M., AND JONES, v. E. 1973. hmUUity in the parasitic relationship between helminths and hosts. Progress in Allergy 17, 93-144. ROTHWELL, T. L. W., AND DINEEN, J. K. 1972. Cellular reactions in guinea pigs following primary and challenged infection with Trichostrongylus colubriformis with special reference to the roles played by eosinophils and basophils in rejection of the parasite. Immunology 22, 733-745. STOLL, N. R. 1929. Studies with the strongylid nematode, Haemonchus contortus. I. Acquired resistance ‘of hosts under natural reinfection conditions out-of-doors. American Journal of Hygiene 10,384-4X
CHARACTERISTICS
IN
MICE
43
TALKFERRO, W. H., AND SARLES, M. P.1939. The cellular reactions in the skin, lungs and intestine of normal and immune rats after infection with Nippostrongylus muris. .lournul of Infectious Diseases 64, 157-162. VAN ZANDT, P. D. 1961. Studies on the immunity relationships in white mice given infections with Nematospiroides dubius Baylis, 1926 (Nematoda: Heligmosomidae) . Journal of the Elisha Mitchell Science So&@ 77, 300-309. WAKELIN, D. 1967. Acquired immunity to Trichuris mu& in the albino laboratory mouse. Parasitology 57, 515-524. WELLS, I’. D. 1962. Mast cell, eosinophil and histamine levels in Nippostrongylus brasiliensis infected rats. Experimental Parasitology 12, 82. ZIDIAN, J. L. 1975. The influence of host genetic strain on resistance to Heligmosomoides polygyrus in the mouse. Masters thesis. University of Pittsburgh.
