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Ascaris suum: Immunization with soluble antigens in the guinea pig
Inrernational Journal for Parasilolo.qy. 1977.Vol. 7. pp. 287-291. Pergamon Press. Prinfed in Great Britain.
ASCARIS SUUM: IMMUNIZATION WITH SOLUBLE ANTIGENS IN THE GUINEA PIG B. E. STROMBERG and E. J. L. SOULSBY Department
of Pathobiology, Laboratory of Parasitology, School of Veterinary Medicine Hl University of Pennsylvania, Philadelphia, PA 19174, U.S.A. (Received 29 November
,
1976)
B. E. and SOULSBY E. J. L. 1977. Ascavis suum: immunization with soluble antigens in the guinea pig. International Journalfor Parasitology 7: 287-291. The capacity of extracts or excretory-secretory products of adult and larval stages of Ascaris suum to induce protective immunity was evaluated. Extracts of 2nd, 3rd or 4th-stage larvae were unable to induce significant protection while adult worms did so. The excretory-secretory products of the adult or larval stages did not induce protection to a challenge infection. However, larvae cultured from the 3rd to the 4th stage produced a substance which was capable of inducing a significant level of protection. It is postulated that this immunogen is associated with the metabolism of the larvae and is involved with the developmental and moulting processes.
AbStrRCt-STROMBERG
INDEX KEY WORDS: immunization.
Ascaris suum; guinea pig; soluble antigens; excretory-secretory
animals were housed in cages with wire mesh floors and fed on a balanced commercial preparation and water,
INTRODUCTION
ad lib. Preparation
of extracts. Adults of A. suum were collected from the small intestine of recently slaughtered swine at a local abattoir. They were washed in cold running tap water for 30 min, allowed to drain and placed in a Waring Blender with an equal part (w:v) of 0.1 MTris-HCI buffer, pH 7.2 and homogenized at low speed for 1 min. The homogenate was placed in a beaker and allowed to extract for 18 h at 4°C. The insoluble material was removed by filtration through a 40 mesh sieve, centrifuged at 8800 g for 30 min and then stored at -20°C. Infective (2nd~stage) larvae were obtained by artificially hatching embryonated eggs as previously described (Stromberg & Soulsby, 1976). They were washed in 0.15 M-phosphate buffered saline (PBS), pH 7.2, disrupted by sonication and extracted for 18 h at 4°C. The insoluble material was removed by centrifugation at 8800 g for 30 min and the supernatant fluid stored at -20°C prior to use. Third-stage larvae were obtained from the lungs of rabbits 7 days after oral infection with 100,000 embryonated eggs of A. suum by the Baermann technique. Fourth-stage larvae were obtained by culturing 3rd-stage larvae through the 3rd moult to the 4th stage (Sylk, Stromberg & Soulsby, 1974). Extracts of 3rd and 4th larval stages were prepared by grinding in a Ten-Broeck glass tissue homogenizer in PBS and extracting the material for 18 h at 4°C. The insoluble material was removed by centrifugation and stored as above. Preparation of excretory-secretory products. Adult A. SUUWI were obtained from the intestine of recently slaughtered swine at the local abattoir and were collected into warm (37°C) 0.15 M-NaCl (saline) containing antibiotics (penicillin, 300 units; streptomycin, 0.3 mg;
investigators have reported limited success in inducing resistance to an Ascaris suum infection with extracts of adult worms (Fallis, 1948; Berger & Wood, 1964; Leikina, 1965; Bindseil, 1969) while others were unable to demonstrate any protection (Martin, 1926; Sprent & Chen, 1949). Some investigators have reported variable success using extracts of larval stages (Soulsby, 1963; Crandall & Arean, 1965; Lehnert, 1967). Immunization with the excretory-secretory (ES) products of various larval stages has also produced limited protection against challenge infection. However, the use of different antigens and techniques in these reports does not allow comparison to determine their relative effectiveness. This study evaluates the ability of soluble antigens to induce immunity to a challenge infection of A. suum. Extracts of adult worms or infective, 3rd or 4th-stage larvae or the ES products produced by culturing adults, infective, 3rd or 4th-stage larvae or 3rd-stage larvae cultured through the 3rd moult to the 4th stage were administered and the degree of protection determined. SEVERAL
MATERIALS Experimental
products;
AND METHODS
animals. Randomly
bred female Hartley strain guinea pigs (550 g) were used throughout the experiments. Female New Zealand white rabbits (3 kg) were used to provide third stage larvae of A. SUUM.The 287
B.E.
288
STROMBERG
mycostatin, 100 units/ml) in a thermal insulated flask. In the laboratory they were washed in warm (37°C) running tap water for 30 mitt, purged of intestinal contents in 0.0015 N NaOH for 90 min at 37°C (Wharton, 1915) and placed in a sterilizing solution (penicillin,
1000 units; streptomycin, units/ml) for 30 min, after
1.3 mg;
mycostatin
250
which they were washed in warm sterile saline. They were then incubated at 37°C in tissue culture medium 199 (Grand Island Biological Co., New York), supplemented with 0.0225 mg glucose, 300 units penicillin, 0.3 mg streptomycin and 100 units mycostatin ml, which was previously sterilized by filtration through a 0.45 urn millipore filter. Two hundred worms were cultured in 250 ml of culture medium in a one liter flask, which was stoppered and maintained at 37°C. After 18 h incubation the supernatant fluid was filtered through a 64um aperture Nitex mesh (A. H. Thomas, Philadelphia, PA) to remove the insoluble material and then concentrated 50 times by dialysis against carbowax (Calbiochem, CA). Infective (Znd-stage) larvae were hatched as previously described (Stromberg & Soulsby, 1976). Larvae were sterilized as described above for the adult worms and cultured in a I5 ml conical centrifuge tube containing 50,000 larvae in 10 ml of the basic culture medium used for culturing adult worms. The tubes were closed with rubber stoppers and rotated on a roller drum at 37°C for I8 h. The cultures were pooled and the supernatant fluid was recovered by filtration through 64 urn aperture Nitex mesh and concentrated 20 times on an Amicon filter (Model 52) using a filter membrane (PM 10) with a 10,000 molecular weight exclusion limit. Third-stage larvae were obtained from the lungs of rabbits as detailed above. The larvae were washed, sterilized and cultured in 200 ml French Square bottles at a concentration of 2000 in 20 ml of the basic culture medium. The bottles were closed with rubber stoppers and incubated for 5 days at 37°C on a roller drum. The cultures were pooled and the supernatant recovered and concentrated as described for the infective larvae. The ES products of the 4th-stage larvae were obtained by culturing in vim derived 4th-stage larvae in the basic culture medium. The cultures were pooled, filtered and concentrated as described above, The ES product antigens associated with moulting were collected from cultures in which 3rd-stage larvae were cultured to the 4th stage (Sylk et al., 1974). Two thousand 3rd-stage larvae obtained as described previously were cultured in the basic culture medium supplemented with swine serum (0.04 ml/ml) and an atmosphere of N,: CO,: 0, (90:5:5). After 5 days in culture at least 9504 of the larvae had moulted to the 4th-stage; the culture fluid was collected and prepared as described above. Culture medium controls were prepared and treated identically with the exception that the ascarids were not included.
Ewperirrental rlesign. Experimental groups generally contained six or more guinea pigs in each. Animals were sensitized by a single intramuscular injection of antigen in Freund’s complete adjuvant (Difco) in the muscle of the hind legs. Each immunizing dose was characterized as total protein and the number of larvae or adults needed to produce the antigen (larval equivalents) and are presented in Table 1. For each experimental group one or two control groups were used in which the animals were immunized similarly with control culture medium and/or saline. Ten days after immunization the animals were challenged by the injection of 5000 artifically
and E.J.L.
SOULSBY
I.J.P. VOL.
7. 1977
hatched 2nd-stage larvae into the mesenteric vein as previously described (Stromberg & Soulsby, 1976). Protection was assessed by comparing the percent recovery of larvae obtained from the lungs of experimental animals to control animals 6 days after challenge. The mean percent recovery of 3rd-stage larvae data was transformed with the arc-sin conversion to obtain normally distributed data. All experimental groups were compared with the saline controls using Student’s t test or the Fisher-Behrens modified t test when there was inequality of the sample variances. A probability value (P) of 0.025 or less was considered to constitute a statistically significant difference between groups. The percent protection was calculated as the percent reduction of recovery from the saline control mean. TABLE
~-ADULT OR LARVAL EQUIVALENTS AND PROTEIN CONCENTRATION OF EACH IMMUNIZIh'G DO.%
Antigen
Equivalents Extracts 10 I00,000 2000 2000
Adult L, L:, I.,
Adult L, L, Lr L, -+ L,
Protein
Excretory-secretory 100 50,000 1000
*Determined
2.05 0.75 0.41 0.44 products
IO00 1000 by Biuret
(mg)*
0.78 I.88 0.38 0.25 0.75
reaction.
RESULTS
The results of these experiments are presented in tabular form in Tables 2 and 3. Each table presents the sensitizing antigen, the mean percentage of recovery (plus or minus the standard error of the mean) of 3rd-stage larvae from the lungs, the percent protection and a P value if significant. The results of experiments in which guinea pigs were immunized with various extracts of A, suum are presented in Table 2. Animals immunized with an extract of adult ascarids were significantly protected and showed a 34% reduction in larval recovery. All other extracts failed to induce a significant level of protection. Data for the animals immunized with the ES products are presented in Table 3. Only the ES products of larvae developing from the third to the fourth stage induced significant protection. Control animals immunized with normal culture medium did not differ significantly from the saline control animals. DISCUSSION The extract of adult whole worms induced significant resistance to a challenge infection. These results concur with those of Leikina (1953, 1965)
I.J.P.VOL.7.
1977
A. SUUM: immunization
in the guinea pig
289
TABLE 2-ANIMALSSENSITIZED WITH EXTRACTS OF Ascarissuum. MEAN PERCENT RECOVERY* OFTHIRD STAGE LARVAE 0F Ascaris swum FROM THE LUNGS OF GUINEA PIGS SENSITIZED By INJECTION OF DIFFERENT EXTRACTS OF A. suum AND CHALLENGED WITH 5000SECOND STAGE LARVAEOF A.suum BY MESENTERIC VElNINJECTION
Experiment I
II 111 IV
Group 1 2 1 2 1 2
Sensitizing extract Adult Saline L Saline L Saline
I
L
2
Saline
Mean & S.E.I_ 10.00 15.16 16.69 19.68 18.23 17.27 17.94 17.27
f i $ i & f i i
0.80 0.85 0.74 3.22 1.57 0.53 0.62 0.53
‘A Protection
P Value
34.02
0.001
15.19
NS$
- -5.56
NSQ:
- 3.88
NS
*Arc-Sin transformed data. tS.E. = Standard error of the mean. @Fisher-Behrens modified t test.
TABLE ~-ANIMALS SENSITIZED WITH EXCRETORY-SECRETORY PRODUCTS OF Ascaris SUUM. MEAN PERCENT RECOVERY* OF THIRD STAGE LARVAE OF Ascaris sl.iumFROM THE LUNGS OF GUINEA PIGS SENSITIZED BY INJECTION OF DIFFERENT EXCRETORY-SECRETORY PRODUCTS OF A. SNNm AND CHALLENGED WITH 5000 SECOND STAGE LARVAE OF A. SUllm BY MESENTERIC VEIN INJECTION
Experiment
1
Group 1 2 3
II 111 1v
V
1 2 3 1 2 3 1 2 3
I 2 3
Sensitizing ES product Adult Saline Mediumt L Saline Medium f-3 Saline Medium l-4 Saline Medium l-3 - L Saline Medium
Mean S.E.? 12.99 14.69 13.35 19.70 20.30 18.33 16.97 16.85 17.22 17.28 19.65 17.92 10.67 20.02 19.49
& & f + * i + i * $ i * i f i
0.79 1.47 1.34 2.23 2.19 1.61 0.96 2.07 172 1.50 3.20 1.92 0.90 2.14 1.44
% Protection
P Value
11.57
NS
2.96
NS
-0.71
NSS
12.03
NS
46.72
o.oosg
*Arc-Sin transformed data. 7S.E. = Standard error of the mean. $Medium = Culture medium control. §Fisher-Behrens modified I test. who reported strong protection against a challenge infection. Other investigators have been able to induce little protection using extracts of adult ascarids (Kerr, 1938; Fallis, 1944; Berger & Wood, 1964; Bindseil, 1969). The significant degree of protection of this experiment, in view of the limited success of other investigators, may be due to the quantitative techniques used in this study or to the relatively high protein concentration of the extract. The dose response to whole worm extract is presently under consideration. The extract of infective larvae of A. suum was unable to induce a statistically significant level of
protection. This confirms the work of Crandall & Arean (1965) and that of Guerrero & Silverman (1969) who also observed that somatic antigens of 2nd-stage larvae failed to induce resistance to a challenge infection. The extract of 3rd-stage larvae was also unable to induce significant protection. These results differ with those of Guerrero & Silverman (1969) in which it was demonstrated that somatic antigens of 3rdstage larvae induced about 59”/0 protection in mice, which was significant at the 0.05 level. Guerrero & Silverman (1971) also reported some success with somatic antigens of 3rd- and 4th-
290
B.E. STROMBERG and E. J. L. SOLJLSBY
larval stages. The present experiments did not demonstrate any protection with the extract of the 4th larval stage. The ES products of adult A. suum produced in culture were unable to induce significant protection. This would indicate that the adult worms are not an effective source of antigens in viva. An important source of ‘protective’ antigens has been considered to be the ES antigens of infective or 2nd~stage larvae (Soulsby, 1957). This concept was supported by Crandall & Arean (1965) who were able to protect mice against a challenge infection using the ES products of infective larvae. They were also able to immunize animals by placing living infective larvae in millipore chambers into the peritoneal cavity. Guerrero & Silverman (1969) were unable to induce protective immunity in mice with the ES products derived from 5 days in vitro cultures of 2nd-stage larvae, however w-hen the larvae were cultured 12 days the ES antigens (referred to as metabolic antigens of late 2nd- and early 3rd-stage larvae) were able to induce some protection (Guerrero & Silverman, 1971). In these experiments the ES products of 2nd-stage larvae were unable to induce resistance to a challenge infection. The ES products of 3rd~stage larvae were also incapable of inducing resistance. Soulsby (1963) reported protection in the guinea pig using the supernatant from cultures of 3rd~stage larvae. Likewise, Guerrero & Silverman (1969, 1971) were able to produce resistance in mice with the culture media in which 3rd~stage larvae were maintained for 5 days. The authors refer to these ES products as those produced by late 3rd and early 4th-stage larvae and one must therefore assume that some larval development must have taken place in the culture. In this study the ES products were obtained from 3rd-stage larvae maintained in culture for 5 days during which time there was no development to the 4th-stage according to the descriptions of Nichols (1956) and Douvres, Tromba & Matakatis (1969). The lack of development of infective, 3rd- or 4th~stage larvae in culture may account for the lack of success in producing resistance to a challenge infection. The use of a single sensitizing injection may also account for poor protection when compared with the success of multiple injections used by the other investigators. The ES products of 3rd~stage larvae which had developed to the 4th~stage were able to induce significant resistance to a challenge infection. This would suggest that protective antigens are produced during a period when larvae are actively developing rather than being maintained in culture. This protective antigen might be related to the moulting process. Soulsby (1959) suggested that the moultingperiod of 2nd~stage larvae of A. suztm in the guinea pig was the first time that significant amounts of
I.J.P. VQL.7. 1977
antigen were released during the infection. Guerrero & Silverman (1969) concluded that a marked release of metabolic antigen occurs between the 3rd and 4th larval stages of A. suum. The importance of the moulting period of larval nematodes has been demonstrated in the ‘self cure’ reaction in Haemonthus contorrus infection. Thus Soulsby, Sommerville & Stewart (1959) and Soulsby & Stewart (1960) demonstrated that it was during the moulting period that the immediate hypersensitivity reaction was initiated which caused the adults of H. contortus to be expelled. Acknowiedgements-The
authors wish to express their to Mr. Derek Muncey, Mrs. Rosetta Goss and Miss Norma Molina for their assistance throughout these studies. This work was supported in part by appreciation
U.S.P.H.S. Research Grant AI-06262, Research Training Grant AI-00302 and Biomedial Sciences Support Grant RRO-7083. REFERENCES BERCER H. & WOOD T. 3. 1964. Immunological studies with Ascaris suum in rabbits with observations on natural and artificially acquired immunity. Journal of Parasitology 50: Suppl. 25 -26 (Abstr.). BINDSEIL E. 1969. Immunity to Ascnris suutr-1. Im-
munity induced in mice by means of material from adult worms. Acta Pathologica et Microbiologica Scandin~ica 77: 223-234. CRANDALL C. A. & AREAN V. M. 1965. The protective effect of viable and non-viable Ascaris suum larvae and egg preparations in mice. American Journal nf Tropical Medicine and Hygiene 14: 765-769. DOUVRES F. W., TROMBAF. G. & MALAKATISC. M. 1969. Morphogenesis and migration of Ascaris suum larvae developing to the fourth stage in swine. Jffffrnul uf Parusitoiogy 55: 689-7 12 FALLIS A. M. 1944. Resistance to Ascaris lumbricoides
infection as demonstrated experimentally in guinea oies. Canadian Journal of Public Health 35: 90. FALLIS A. M. 1948. As&is iu~brieoides infection in guinea pigs with special reference to eosinophiiia and resistance, Canadian Journa! of Research D26: 307-327. GUERREROJ. & SILVERMANP. H. 1969. Ascaris suum. Immune reactions in mice-l. Larval metabolic and somatic antigens. Experitnentaf Parasitology 26: 272-281.
GUERREROJ. & SILVERMANP. H. 1971. Ascaris suum: immune reactions in mice--II. Metabolic and somatic antigens of in vitro cultured larvae. Experimental Parasitology 29: 1l&l 15. KERR K. B. 1938. Studies on the passive transference of acquired resistance to dog hookworm and the pig ascaris. American Journal of hygiene 27: 60-66. LEHNERTJ. P. 1967. Studies on the biology and immunology of Ascuvis suum in mice. Ph.D. Thesis. University of Illinois. LEIKINA E. S. 1953. Early immunological diagnosis of ascaridosis. In Contributions to Heltninihology, pp. 362-371. Academy of Science, U.S.S.R., Moscow. LE~KINAE. S. $965. Research on ascariasis immunity and immunodiagnosis. Bulletin of the World Health Organization 32: 699-708.
I.J.P. VOL.7. 1977
A. suum: immunization
MARTINH. M. 1926. Studies on the Ascaris lumbricoides. Research Bulletin of the Nebraska Agricultural Experiment Station 37: l-78. NICHOLS R. L. 1956. The etiology of visceral larva migrans-Il. Comparative larval morphology of Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis and Ancylostoma caninum. Journal of Parasitology 42: 363-399. SOULSBYE. J. L. 1957. Immunization against Ascaris lumbricoides in the guinea pig. Nature (Lond.) 179: 783-784. SOULSBYE. J. L. 1959. The importance of the moulting period in the stimulation of immunity to helminths. XVI International Veterinary Congress, Madrid 2: Y-573. SOULSBYE. J. L. 1963. The nature and origin of the functional antigens in helminth infections. Annals of the New York Academy of Science 113: 492-509. SOULSBYE. J. L. & STEWARTD. F. 1960. Serological studies of the self-cure reaction in sheep infected with Haemonchus contortus. Australian Journal of Agricultural Research 11: 595-603.
in the guinea pig
291
SOULSBYE. J. L., SOMMERVILLE R. I. & STEWARTD. F. 1959. Antigenic stimulus of exsheathing fluid in the self-cure of sheep infected with Haemonchus confortus. Nature (Lond.) 183: 553-554. SPRENT J. F. A. & CHEN H. H. 1949. Immunological studies in mice infected with the larvae of Ascaris lumbricoides-I. Criteria of immunity and immunizing effect of isolated worm tissues. Journal of Inj&tious Disease 84: 111-124. STROMBERG B. E. & SOULSBYE. J. L. 1976. The protective effect of developmental stages in an Ascaris suum infection in guinea pigs. Veterinary Parasitology 2: 197-208. SYLK S. R., STROMBERG B. E. & SOULSBYE. J. L. 1974. Development of Ascaris suum larvae from the third to the fourth stage, in vitro. International Journal for Parasirology 4: 261-265. WHARTONL. D. 1915. The development of the eggs of Ascaris lumbricoides. Philippine Journal of Science, Manila, Section B-Tropical Medicine 10: 19-23.
ASCARIS SUUM: IMMUNIZATION WITH SOLUBLE ANTIGENS IN THE GUINEA PIG B. E. STROMBERG and E. J. L. SOULSBY Department
of Pathobiology, Laboratory of Parasitology, School of Veterinary Medicine Hl University of Pennsylvania, Philadelphia, PA 19174, U.S.A. (Received 29 November
,
1976)
B. E. and SOULSBY E. J. L. 1977. Ascavis suum: immunization with soluble antigens in the guinea pig. International Journalfor Parasitology 7: 287-291. The capacity of extracts or excretory-secretory products of adult and larval stages of Ascaris suum to induce protective immunity was evaluated. Extracts of 2nd, 3rd or 4th-stage larvae were unable to induce significant protection while adult worms did so. The excretory-secretory products of the adult or larval stages did not induce protection to a challenge infection. However, larvae cultured from the 3rd to the 4th stage produced a substance which was capable of inducing a significant level of protection. It is postulated that this immunogen is associated with the metabolism of the larvae and is involved with the developmental and moulting processes.
AbStrRCt-STROMBERG
INDEX KEY WORDS: immunization.
Ascaris suum; guinea pig; soluble antigens; excretory-secretory
animals were housed in cages with wire mesh floors and fed on a balanced commercial preparation and water,
INTRODUCTION
ad lib. Preparation
of extracts. Adults of A. suum were collected from the small intestine of recently slaughtered swine at a local abattoir. They were washed in cold running tap water for 30 min, allowed to drain and placed in a Waring Blender with an equal part (w:v) of 0.1 MTris-HCI buffer, pH 7.2 and homogenized at low speed for 1 min. The homogenate was placed in a beaker and allowed to extract for 18 h at 4°C. The insoluble material was removed by filtration through a 40 mesh sieve, centrifuged at 8800 g for 30 min and then stored at -20°C. Infective (2nd~stage) larvae were obtained by artificially hatching embryonated eggs as previously described (Stromberg & Soulsby, 1976). They were washed in 0.15 M-phosphate buffered saline (PBS), pH 7.2, disrupted by sonication and extracted for 18 h at 4°C. The insoluble material was removed by centrifugation at 8800 g for 30 min and the supernatant fluid stored at -20°C prior to use. Third-stage larvae were obtained from the lungs of rabbits 7 days after oral infection with 100,000 embryonated eggs of A. suum by the Baermann technique. Fourth-stage larvae were obtained by culturing 3rd-stage larvae through the 3rd moult to the 4th stage (Sylk, Stromberg & Soulsby, 1974). Extracts of 3rd and 4th larval stages were prepared by grinding in a Ten-Broeck glass tissue homogenizer in PBS and extracting the material for 18 h at 4°C. The insoluble material was removed by centrifugation and stored as above. Preparation of excretory-secretory products. Adult A. SUUWI were obtained from the intestine of recently slaughtered swine at the local abattoir and were collected into warm (37°C) 0.15 M-NaCl (saline) containing antibiotics (penicillin, 300 units; streptomycin, 0.3 mg;
investigators have reported limited success in inducing resistance to an Ascaris suum infection with extracts of adult worms (Fallis, 1948; Berger & Wood, 1964; Leikina, 1965; Bindseil, 1969) while others were unable to demonstrate any protection (Martin, 1926; Sprent & Chen, 1949). Some investigators have reported variable success using extracts of larval stages (Soulsby, 1963; Crandall & Arean, 1965; Lehnert, 1967). Immunization with the excretory-secretory (ES) products of various larval stages has also produced limited protection against challenge infection. However, the use of different antigens and techniques in these reports does not allow comparison to determine their relative effectiveness. This study evaluates the ability of soluble antigens to induce immunity to a challenge infection of A. suum. Extracts of adult worms or infective, 3rd or 4th-stage larvae or the ES products produced by culturing adults, infective, 3rd or 4th-stage larvae or 3rd-stage larvae cultured through the 3rd moult to the 4th stage were administered and the degree of protection determined. SEVERAL
MATERIALS Experimental
products;
AND METHODS
animals. Randomly
bred female Hartley strain guinea pigs (550 g) were used throughout the experiments. Female New Zealand white rabbits (3 kg) were used to provide third stage larvae of A. SUUM.The 287
B.E.
288
STROMBERG
mycostatin, 100 units/ml) in a thermal insulated flask. In the laboratory they were washed in warm (37°C) running tap water for 30 mitt, purged of intestinal contents in 0.0015 N NaOH for 90 min at 37°C (Wharton, 1915) and placed in a sterilizing solution (penicillin,
1000 units; streptomycin, units/ml) for 30 min, after
1.3 mg;
mycostatin
250
which they were washed in warm sterile saline. They were then incubated at 37°C in tissue culture medium 199 (Grand Island Biological Co., New York), supplemented with 0.0225 mg glucose, 300 units penicillin, 0.3 mg streptomycin and 100 units mycostatin ml, which was previously sterilized by filtration through a 0.45 urn millipore filter. Two hundred worms were cultured in 250 ml of culture medium in a one liter flask, which was stoppered and maintained at 37°C. After 18 h incubation the supernatant fluid was filtered through a 64um aperture Nitex mesh (A. H. Thomas, Philadelphia, PA) to remove the insoluble material and then concentrated 50 times by dialysis against carbowax (Calbiochem, CA). Infective (Znd-stage) larvae were hatched as previously described (Stromberg & Soulsby, 1976). Larvae were sterilized as described above for the adult worms and cultured in a I5 ml conical centrifuge tube containing 50,000 larvae in 10 ml of the basic culture medium used for culturing adult worms. The tubes were closed with rubber stoppers and rotated on a roller drum at 37°C for I8 h. The cultures were pooled and the supernatant fluid was recovered by filtration through 64 urn aperture Nitex mesh and concentrated 20 times on an Amicon filter (Model 52) using a filter membrane (PM 10) with a 10,000 molecular weight exclusion limit. Third-stage larvae were obtained from the lungs of rabbits as detailed above. The larvae were washed, sterilized and cultured in 200 ml French Square bottles at a concentration of 2000 in 20 ml of the basic culture medium. The bottles were closed with rubber stoppers and incubated for 5 days at 37°C on a roller drum. The cultures were pooled and the supernatant recovered and concentrated as described for the infective larvae. The ES products of the 4th-stage larvae were obtained by culturing in vim derived 4th-stage larvae in the basic culture medium. The cultures were pooled, filtered and concentrated as described above, The ES product antigens associated with moulting were collected from cultures in which 3rd-stage larvae were cultured to the 4th stage (Sylk et al., 1974). Two thousand 3rd-stage larvae obtained as described previously were cultured in the basic culture medium supplemented with swine serum (0.04 ml/ml) and an atmosphere of N,: CO,: 0, (90:5:5). After 5 days in culture at least 9504 of the larvae had moulted to the 4th-stage; the culture fluid was collected and prepared as described above. Culture medium controls were prepared and treated identically with the exception that the ascarids were not included.
Ewperirrental rlesign. Experimental groups generally contained six or more guinea pigs in each. Animals were sensitized by a single intramuscular injection of antigen in Freund’s complete adjuvant (Difco) in the muscle of the hind legs. Each immunizing dose was characterized as total protein and the number of larvae or adults needed to produce the antigen (larval equivalents) and are presented in Table 1. For each experimental group one or two control groups were used in which the animals were immunized similarly with control culture medium and/or saline. Ten days after immunization the animals were challenged by the injection of 5000 artifically
and E.J.L.
SOULSBY
I.J.P. VOL.
7. 1977
hatched 2nd-stage larvae into the mesenteric vein as previously described (Stromberg & Soulsby, 1976). Protection was assessed by comparing the percent recovery of larvae obtained from the lungs of experimental animals to control animals 6 days after challenge. The mean percent recovery of 3rd-stage larvae data was transformed with the arc-sin conversion to obtain normally distributed data. All experimental groups were compared with the saline controls using Student’s t test or the Fisher-Behrens modified t test when there was inequality of the sample variances. A probability value (P) of 0.025 or less was considered to constitute a statistically significant difference between groups. The percent protection was calculated as the percent reduction of recovery from the saline control mean. TABLE
~-ADULT OR LARVAL EQUIVALENTS AND PROTEIN CONCENTRATION OF EACH IMMUNIZIh'G DO.%
Antigen
Equivalents Extracts 10 I00,000 2000 2000
Adult L, L:, I.,
Adult L, L, Lr L, -+ L,
Protein
Excretory-secretory 100 50,000 1000
*Determined
2.05 0.75 0.41 0.44 products
IO00 1000 by Biuret
(mg)*
0.78 I.88 0.38 0.25 0.75
reaction.
RESULTS
The results of these experiments are presented in tabular form in Tables 2 and 3. Each table presents the sensitizing antigen, the mean percentage of recovery (plus or minus the standard error of the mean) of 3rd-stage larvae from the lungs, the percent protection and a P value if significant. The results of experiments in which guinea pigs were immunized with various extracts of A, suum are presented in Table 2. Animals immunized with an extract of adult ascarids were significantly protected and showed a 34% reduction in larval recovery. All other extracts failed to induce a significant level of protection. Data for the animals immunized with the ES products are presented in Table 3. Only the ES products of larvae developing from the third to the fourth stage induced significant protection. Control animals immunized with normal culture medium did not differ significantly from the saline control animals. DISCUSSION The extract of adult whole worms induced significant resistance to a challenge infection. These results concur with those of Leikina (1953, 1965)
I.J.P.VOL.7.
1977
A. SUUM: immunization
in the guinea pig
289
TABLE 2-ANIMALSSENSITIZED WITH EXTRACTS OF Ascarissuum. MEAN PERCENT RECOVERY* OFTHIRD STAGE LARVAE 0F Ascaris swum FROM THE LUNGS OF GUINEA PIGS SENSITIZED By INJECTION OF DIFFERENT EXTRACTS OF A. suum AND CHALLENGED WITH 5000SECOND STAGE LARVAEOF A.suum BY MESENTERIC VElNINJECTION
Experiment I
II 111 IV
Group 1 2 1 2 1 2
Sensitizing extract Adult Saline L Saline L Saline
I
L
2
Saline
Mean & S.E.I_ 10.00 15.16 16.69 19.68 18.23 17.27 17.94 17.27
f i $ i & f i i
0.80 0.85 0.74 3.22 1.57 0.53 0.62 0.53
‘A Protection
P Value
34.02
0.001
15.19
NS$
- -5.56
NSQ:
- 3.88
NS
*Arc-Sin transformed data. tS.E. = Standard error of the mean. @Fisher-Behrens modified t test.
TABLE ~-ANIMALS SENSITIZED WITH EXCRETORY-SECRETORY PRODUCTS OF Ascaris SUUM. MEAN PERCENT RECOVERY* OF THIRD STAGE LARVAE OF Ascaris sl.iumFROM THE LUNGS OF GUINEA PIGS SENSITIZED BY INJECTION OF DIFFERENT EXCRETORY-SECRETORY PRODUCTS OF A. SNNm AND CHALLENGED WITH 5000 SECOND STAGE LARVAE OF A. SUllm BY MESENTERIC VEIN INJECTION
Experiment
1
Group 1 2 3
II 111 1v
V
1 2 3 1 2 3 1 2 3
I 2 3
Sensitizing ES product Adult Saline Mediumt L Saline Medium f-3 Saline Medium l-4 Saline Medium l-3 - L Saline Medium
Mean S.E.? 12.99 14.69 13.35 19.70 20.30 18.33 16.97 16.85 17.22 17.28 19.65 17.92 10.67 20.02 19.49
& & f + * i + i * $ i * i f i
0.79 1.47 1.34 2.23 2.19 1.61 0.96 2.07 172 1.50 3.20 1.92 0.90 2.14 1.44
% Protection
P Value
11.57
NS
2.96
NS
-0.71
NSS
12.03
NS
46.72
o.oosg
*Arc-Sin transformed data. 7S.E. = Standard error of the mean. $Medium = Culture medium control. §Fisher-Behrens modified I test. who reported strong protection against a challenge infection. Other investigators have been able to induce little protection using extracts of adult ascarids (Kerr, 1938; Fallis, 1944; Berger & Wood, 1964; Bindseil, 1969). The significant degree of protection of this experiment, in view of the limited success of other investigators, may be due to the quantitative techniques used in this study or to the relatively high protein concentration of the extract. The dose response to whole worm extract is presently under consideration. The extract of infective larvae of A. suum was unable to induce a statistically significant level of
protection. This confirms the work of Crandall & Arean (1965) and that of Guerrero & Silverman (1969) who also observed that somatic antigens of 2nd-stage larvae failed to induce resistance to a challenge infection. The extract of 3rd-stage larvae was also unable to induce significant protection. These results differ with those of Guerrero & Silverman (1969) in which it was demonstrated that somatic antigens of 3rdstage larvae induced about 59”/0 protection in mice, which was significant at the 0.05 level. Guerrero & Silverman (1971) also reported some success with somatic antigens of 3rd- and 4th-
290
B.E. STROMBERG and E. J. L. SOLJLSBY
larval stages. The present experiments did not demonstrate any protection with the extract of the 4th larval stage. The ES products of adult A. suum produced in culture were unable to induce significant protection. This would indicate that the adult worms are not an effective source of antigens in viva. An important source of ‘protective’ antigens has been considered to be the ES antigens of infective or 2nd~stage larvae (Soulsby, 1957). This concept was supported by Crandall & Arean (1965) who were able to protect mice against a challenge infection using the ES products of infective larvae. They were also able to immunize animals by placing living infective larvae in millipore chambers into the peritoneal cavity. Guerrero & Silverman (1969) were unable to induce protective immunity in mice with the ES products derived from 5 days in vitro cultures of 2nd-stage larvae, however w-hen the larvae were cultured 12 days the ES antigens (referred to as metabolic antigens of late 2nd- and early 3rd-stage larvae) were able to induce some protection (Guerrero & Silverman, 1971). In these experiments the ES products of 2nd-stage larvae were unable to induce resistance to a challenge infection. The ES products of 3rd~stage larvae were also incapable of inducing resistance. Soulsby (1963) reported protection in the guinea pig using the supernatant from cultures of 3rd~stage larvae. Likewise, Guerrero & Silverman (1969, 1971) were able to produce resistance in mice with the culture media in which 3rd~stage larvae were maintained for 5 days. The authors refer to these ES products as those produced by late 3rd and early 4th-stage larvae and one must therefore assume that some larval development must have taken place in the culture. In this study the ES products were obtained from 3rd-stage larvae maintained in culture for 5 days during which time there was no development to the 4th-stage according to the descriptions of Nichols (1956) and Douvres, Tromba & Matakatis (1969). The lack of development of infective, 3rd- or 4th~stage larvae in culture may account for the lack of success in producing resistance to a challenge infection. The use of a single sensitizing injection may also account for poor protection when compared with the success of multiple injections used by the other investigators. The ES products of 3rd~stage larvae which had developed to the 4th~stage were able to induce significant resistance to a challenge infection. This would suggest that protective antigens are produced during a period when larvae are actively developing rather than being maintained in culture. This protective antigen might be related to the moulting process. Soulsby (1959) suggested that the moultingperiod of 2nd~stage larvae of A. suztm in the guinea pig was the first time that significant amounts of
I.J.P. VQL.7. 1977
antigen were released during the infection. Guerrero & Silverman (1969) concluded that a marked release of metabolic antigen occurs between the 3rd and 4th larval stages of A. suum. The importance of the moulting period of larval nematodes has been demonstrated in the ‘self cure’ reaction in Haemonthus contorrus infection. Thus Soulsby, Sommerville & Stewart (1959) and Soulsby & Stewart (1960) demonstrated that it was during the moulting period that the immediate hypersensitivity reaction was initiated which caused the adults of H. contortus to be expelled. Acknowiedgements-The
authors wish to express their to Mr. Derek Muncey, Mrs. Rosetta Goss and Miss Norma Molina for their assistance throughout these studies. This work was supported in part by appreciation
U.S.P.H.S. Research Grant AI-06262, Research Training Grant AI-00302 and Biomedial Sciences Support Grant RRO-7083. REFERENCES BERCER H. & WOOD T. 3. 1964. Immunological studies with Ascaris suum in rabbits with observations on natural and artificially acquired immunity. Journal of Parasitology 50: Suppl. 25 -26 (Abstr.). BINDSEIL E. 1969. Immunity to Ascnris suutr-1. Im-
munity induced in mice by means of material from adult worms. Acta Pathologica et Microbiologica Scandin~ica 77: 223-234. CRANDALL C. A. & AREAN V. M. 1965. The protective effect of viable and non-viable Ascaris suum larvae and egg preparations in mice. American Journal nf Tropical Medicine and Hygiene 14: 765-769. DOUVRES F. W., TROMBAF. G. & MALAKATISC. M. 1969. Morphogenesis and migration of Ascaris suum larvae developing to the fourth stage in swine. Jffffrnul uf Parusitoiogy 55: 689-7 12 FALLIS A. M. 1944. Resistance to Ascaris lumbricoides
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GUERREROJ. & SILVERMANP. H. 1971. Ascaris suum: immune reactions in mice--II. Metabolic and somatic antigens of in vitro cultured larvae. Experimental Parasitology 29: 1l&l 15. KERR K. B. 1938. Studies on the passive transference of acquired resistance to dog hookworm and the pig ascaris. American Journal of hygiene 27: 60-66. LEHNERTJ. P. 1967. Studies on the biology and immunology of Ascuvis suum in mice. Ph.D. Thesis. University of Illinois. LEIKINA E. S. 1953. Early immunological diagnosis of ascaridosis. In Contributions to Heltninihology, pp. 362-371. Academy of Science, U.S.S.R., Moscow. LE~KINAE. S. $965. Research on ascariasis immunity and immunodiagnosis. Bulletin of the World Health Organization 32: 699-708.
I.J.P. VOL.7. 1977
A. suum: immunization
MARTINH. M. 1926. Studies on the Ascaris lumbricoides. Research Bulletin of the Nebraska Agricultural Experiment Station 37: l-78. NICHOLS R. L. 1956. The etiology of visceral larva migrans-Il. Comparative larval morphology of Ascaris lumbricoides, Necator americanus, Strongyloides stercoralis and Ancylostoma caninum. Journal of Parasitology 42: 363-399. SOULSBYE. J. L. 1957. Immunization against Ascaris lumbricoides in the guinea pig. Nature (Lond.) 179: 783-784. SOULSBYE. J. L. 1959. The importance of the moulting period in the stimulation of immunity to helminths. XVI International Veterinary Congress, Madrid 2: Y-573. SOULSBYE. J. L. 1963. The nature and origin of the functional antigens in helminth infections. Annals of the New York Academy of Science 113: 492-509. SOULSBYE. J. L. & STEWARTD. F. 1960. Serological studies of the self-cure reaction in sheep infected with Haemonchus contortus. Australian Journal of Agricultural Research 11: 595-603.
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