Intemofional Journal
for Parasitology.
1975. Vol. 5. pp. 427430.
Pergamon Press. Printed in Great Britain.
FAILURE TO VACCINATE LAMBS AGAINST HAEMONCHUS CONTORTUS WITH FUNCTIONAL METABOLIC ANTIGENS IDENTIFIED BY IMMUNOELECTROPHORESIS Department
J. T. M. NEILSON of Veterinary Science, University of Florida, Gainesville, Florida 32611, U.S.A. (Received 19 August 1974)
Abstract-NEILsoN
J. T. M. 1975. Failure to vaccinate lambs against Haemonchus contortus with functional metabolic antigens identified by immunoelectrophoresis. Znfernational Journal for Pamsitology 5: 427-430. Metabolic products from Haemonchus contortus larvae cultured in vitro from the infective third to fourth stage were collected and concentrated. Chromatographic and immunoelectrophoretic analyses were made to study the numbers and activity of antigens in the metabolic products derived from the in vitro cultured larvae. Three-month-old lambs were given a series of injections of metabolic antigens with and without adjuvant at dose rates of 0.05, 0.5 and 5.0 mg antigen protein per injection. These animals and saline injected controls were each challenged with 3000 H. contortus infective larvae after the last antigen injection and killed 35 days later. No difference was seen in the faecal worm egg counts or the differential worm counts among the vaccinated and control animals. The antigen preparation of worm metabolic products conferred no resistance to challenge infection with the parasite. INDEX KEY WORDS: Huemonchus contorfus; lambs; metabolic antigens; immunoelectrophoresis; vaccination.
INTRODUCTION EFFORTS to vaccinate
animals with somatic extracts of nematodes have been largely unsuccessful. The antigens which induce protective immunity are probably produced only in small amounts by the living parasite and appear to be primarily associated with developing larval stages rather than adult worms (Silverman, 1970). If the host is inoculated with antigens prepared from dead worms, the functional antigens are not present in sufficient quantity, are destroyed during the extraction procedure or are concealed by the large amount of nonfunctional foreign material made available to the host’s reticuloendothelial system (Sinclair, 1970). Christie & Brambell (1966) induced a strong resistance to challenge infection with H. contortus in 2-3-month-old lambs by first exposing them to several doses of H. contortus infective larvae followed by anthelmintic treatment which destroyed the larvae before they reached the adult stage. Silverman (1965) obtained protection against H. contortus in 4-6-month-old lambs with two vaccines. One vaccine was prepared from third and fourth stage larvae somatic and metabolic antigens and the other from fourth stage larvae somatic and metabolic antigens which, when administered to lambs, gave 50 and 64 per cent reduction in challenge worm burdens respectively. In the present work, an attempt was made to
identify functional antigens in the metabolic products released by third stage H. contortus larvae following exsheathment and ecdysis to the fourth stage. Functional antigens were defined by the criteria proposed by Dineen (1963). The concentrated metabolic antigens derived from in vitro culture of H. contortus larvae were injected into lambs in an attempt to stimulate resistance to challenge with this parasite. MATERIALS
AND METHODS
H. contortus infective stage larvae were cultured to the fourth stage in vitro according to the method of Neilson (1969). Under these conditions over 90 per cent of the larvae exsheath within 48 h in culture and 70 per cent of these complete the moult to the fourth stage after 72 h. The culture medium does not contain macromolecules. This facilitates the identification and isolation of macromolecular excretions and secretions or metabolic productsliberated into the culture medium by the developing parasite (Neilson, 1969). In addition, Neilson (1969) described the fractionation of the parasite metabolic products by Sephadex gel filtration and polyacrylamide gel electrophoresis. Antisera to the unfractionated metabolic products were produced in rabbits and sheep. Three rabbits, two months of age at the start of the experiments were each injected intramuscularly with 1.0 ml metabolic products (0.5 mg protein) plus 1.0 ml Freund’s complete adjuvant. Thirty days later, 1.0 ml of metabolic products plus 427
428
J. T. M.
1.0 ml adjuvant were injected into the footpads. Fourteen days later, this was followed by a series of three intradermal injections of metabolic products (1.0 ml/injection) at weekly intervals. The rabbits were bled 1 week after the final injection and the serum from each animal pooled. Three sheep, 3 months of age at the start of the experiments and reared parasite free, were each given two intramuscular injections of 10 ml metabolic products (5.0 mg protein) plus 10 ml Freund’s complete adjuvant 30 days apart. After 14 days, this was followed by a series of three intradermal injections of metabolic products (10 ml/injection) at weekly intervals. The sheep were bled one week after the final injection and the serum from each animal pooled. Immunoelectrophoresis of the metabolic products and the Sephadex fractions thereof was done on the immunoelectrophoretic equipment supplied by the Gelman Instrument Co.* and the methods used in the entire procedure were those described by the manufacturer. Standard die 71649 (Gehnan Instrument Co.) was used to punch the desired pattern on each agar coated slide. This pattern gave one central well (1 mm dia.) and two parallel troughs (1 mm broad) on each side of the well, which allowed 3.6 mm diffusion distance between the well and each trough. The metabolic products preparation or the Sephadex samples thereof were placed in the central well, electrophoresed, then one trough was charged with sheep anti metabolic products serum while the other trough received rabbit anti metabolic products serum. In this way a direct comparison could be made of the precipitating antibodies in rabbit versus sheep antisera to the parasite metabolic products and Sephadex fractions thereof. Twenty-three lambs approximately 3 months of age, reared under worm free conditions, were distributed into 5 groups. Groups 1, 2, 3 and 4 contained 5 animals per group while group 5 contained 3 animals. Each lamb in groups 1,2 and 3 was injected on each occasion with 0.1, 1.0 and 10 ml (equivalent to 0.05,0.5 and 5.0 mg protein) H. contortus metabolic antigen respectively, according to the following schedule: Two injections intramuscularly with the appropriate volume of Freund’s complete adjuvant on day 1 and day 28 followed by a series of intradermal injections of antigen preparation on days 42, 49, 56 and 63. Group 4 animals were similarly injected with saline, 10 ml with or without adjuvant being administered on each occasion. The lambs in Group 5 were untreated. One week after the final injection, on day 70 of the experiment, all lambs were each infected orally with 3000 H. contortus infective larvae. Faecal worm egg counts were performed by the McMaster technique on 2 g faecal samples. The first faecal sample was collected on day 13 postinfection then at 3 or 4 day intervals thereafter. All animals were killed on day 35 after infection and the total number of adult worms and larval stages in each animal counted. The procedure for the recovery and counting of worms has been described (Dineen, Donald, Wagland & Offner, 1965). RESULTS Tmmunoelectrophoresis * Gelman Instrument Michigan 48106.
of the metabolic
products
Co., P.O. Box 1448, Ann Arbor,
NEILSON
I.J.P. VOL.
from in vitro cultured H. contortus larvae and logous antisera prepared in sheep and rabbits 4 and 7 precipitin arcs respectively (Fig. 1). additional precipitating antibodies had been RaES
m--
-
ES
-0
_‘cl,
SaES
---
ES8
0
SaES
-
RaES
-
ES9 SaES
-
-
ES6
RaES
homoyielded Thus 3 stimu-
0
SaES
RaES
5. 1975
x 0
-
FIG. 1. The major precipitin bands obtained following immunoelectrophoresis of unfractionated metabolic antigens (ES), fraction 2 from Sephadex GlOO column (ES6), and fractions 1 and 2 from Sephadex G200 column (ES8 and ES9 respectively) against rabbit and sheep antisera to ES (RaES and SaES respectively). lated in rabbits. One of the responsible antigens had an anodal distribution and the other two had a cathodal distribution. The antigen with anodal electrophoretic mobility was present in fraction 2 from Sephadex G-100 gel filtration while the two antigens with cathodal electrophoretic mobility were located in fractions 1 and 2 from Sephadex G-200 gel filtration. The elution profiles with fraction numbers obtained following gel filtration of the H. contortus metabolic products on Sephadex G50, GlOO and G200 have been reported (Neilson, 1969). The injection of sheep with metabolic antigens derived from in vitro cultures of H. contortus in which larvae developed from the infective third to the fourth stage, failed to confer resistance to challenge with the parasite. No difference in the pattern of faecal egg counts or the number of adult worms found at necropsy was seen among the sheep injected with any of the three doses of the metabolic rntigen preparation, the saline injected controls or sheep given no injections of any kind. The worm burdens are shown in Table 1. Furthermore, no immature or inhibited larval forms of the parasite were noted in the enzymic digests of the abomasums sf any of the sheep.
I.J.P.
VOL.
5.
1975
TABLE
~-MEAN
WORMS
RECOVERED
Antigens of H. contortus f
S.E. ON DAY
OF 3000
Group No.* 1
2 3 4 5
ADULT
Haemonchus contortus
3.5FOLLOWING
LARVAE
PER
AN INFECTION
LAMB
Worm burden 1600 f 190 2140 f 225 1860 * 325 1133 f 88 1600 & 272
*Groups 1, 2 and 3 injected 6 times (see text for injection schedule) with 0.1, 1.0 and 10 ml (equivalent to 0.05, 0.5 and 5.0 mg antigen protein) respectively. Group 4 was saline injected controls and Group 5 received no injections. DISCUSSION Soulsby, Somerville & Stewart (1959), on the basis of serological evidence, proposed that resistance to H. contortus infection in sheep was probably stimulated by antigens released during exsheathment, the terminal part of the second ecdysis and during the third ecdysis. These and other studies have indicated that probably the most logical and rewarding approach to the development of a helminth vaccine would be to cultivate the worm in vitro through those stages which are known to be potentially most immunogenic and to collect the metabolic products released which supposedly contain the functional antigens (Terry, 1968). This rationale was followed in the present study. H. contortus infective stage larvae were allowed to exsheath, then moult to the fourth stage in vitro in a balanced salt solution. The macromolecular substances released by the larvae were isolated and concentrated by methods which would minimize their denaturation (Neilson, 1969). Dineen (1963) proposed that, by a comparison of the serologic responses evoked by parasite antigens given parenterally to the parasite’s natural host and to unnatural hosts, it would be possible to discriminate between functional and casual antigens. Functional antigens are those which stimulate immune responses which adversely affect the biotic potential of the parasite and as such would be suitable components of a vaccine against that parasite. The comparative serologic responses of sheep and rabbits to parenterally injected somatic antigens of H. contortus larvae and adult worms indicated that both contained common functional antigens (Dineen, 1963). It has been shown by others that a vaccine prepared from somatic and metabolic antigens derived from third and fourth stage H. contortus larvae offered a measure of protection in 4-6-monthold lambs against a challenge infection with this parasite (Silverman, 1965). It is possible that this successful vaccine contained the functional antigens described by Dineen (1963) as well as the functional antigens in the excretions and secretions described in the present paper.
429
No protection was obtained in the present work with lambs injected with three dose levels of metabolic products, despite the fact that this preparation contained serologically indicated functional antigens. These findings would suggest that a combination of somatic and metabolic antigens from H. contortus larvae are essential components for a successful non-living vaccine against this parasite. As stated earlier, however, the metabolic products used here, contained, in addition to the serologically identified functional antigens, several nonfunctional antigens. It is possible that the presence of these nonfunctional antigens in the vaccine blocked or otherwise interfered with the induction of protective responses. In addition, earlier electrophoretic studies of the metabolic products (Neilson, 1969) indicate that several proteins in the preparation are not precipitinogens. Purification of those precipitinogens which are functional antigens should be possible by a two step procedure. First, the metabolic products are absorbed with its homologous sheep antiserum so removing the antigens which stimulate common precipitating antibodies in sheep and rabbits. Second, the antigens which stimulate the additional precipitating antibodies in rabbits can be specifically absorbed from the metabolic products by immunosorption on immunosorbent columns prepared with the homologous rabbits antiserum. Further protection studies against H. contortus in sheep should be undertaken using as vaccines various combinations of functional metabolic antigens and functional somatic antigens identified and purified in the manner described above. Three-month-old lambs were used in the present experiment whereas Silverman (1965) in a similar experiment used 4-6-month-old lambs. Urquhart, McIntyre, Mulligan, Jarrett & Sharp (1963) successfully obtained protection to H. contortus in sheep, 6 months or older, following vaccination with irradiated larvae. No such protection was found in similarly vaccinated lambs less than 6 months of age and these workers concluded that these young lambs may not be sufficiently immunologically mature. However, Christie & Bambell (1966) immunized 2-3-month-old lambs by giving several large doses of H. contortus infective larvae followed by two anthelmintic treatments. This pattern of infection followed by anthelmintic treatment was repeated then the lambs and previously uninfected controls were each given a massive challenge. Good protection against the challenge was obtained in the previously exposed lambs and it is interesting to note that the protection obtained in these 2-3-month-old lambs was greater than that obtained in a similar experiment with 8-month-old lambs (Christie, Brambell & Charleston, 1964). Hence no evidence of immunological immaturity in lambs less than 6 months of age was noted in these studies.
430
J. T. M. NEILSON
Acknowledgement-Part
of this work was done at the McMaster Laboratory, CSIRO, Sydney, Australia. This investigation was supported in part by Research Grant GB-18991 from the National Science Foundation. Florida Agricultural Experimental Stations Journal Series No. 5520.
I.J.P. VOL. 5. 1975
NEIUON J. T. M. 1969. Gel filtration and disc electrophoresis of a somatic extract and excretions and secretions of Haemonchus contortus larvae. Experimental Parasitology 25: 131-141. SILVERMAN P. H. 1965. Some immunologic aspects of parasitic helminth infections. American Zoologist 5:
153-163. REFERENCES CHRISTIE M. G. & BRAMBELL M. R. 1966. Acquired resistance to Haemonchus contortus in young lambs.
Journal of Comparative Pathology 76: 207-216. CHRISTIE M. G., BRAMBELL M. R. & CHARLESTON W. A. G. 1964. Resistance to a challenge infection with Haemonchus contortus conferred by previous experience to immature stages only. Journal of Com-
parative Pathology 74: 427434. DINEEN J. K. 1963. Antigenic relationship between host and parasite. Nature 197: 471472. DINEEN J. K., DONALD A. D., WAGLAND B. M. & OFFNER J. 1965. The dynamics of the host-parasite relationship. III. The response of sheep to primary infection with Haemonchus contortus. Parasitology 55:
515-525.
SILVERMAN P. H. 1970. Vaccination: Progress and problems. In Immunitv to Parasitic Animals (Edited bv JACKSON G. J.). Appleton-Century-Crofts, hew York. SINCLAIR I. J. 1970. The relationship between circulaticg antibodies and immunity to helminthic infections.
Advances in Parasitology 8: 97-138. SOULSBY E. J. L., SOMERVILLE R. I. & STEWART D. F. 1969. Antigenic stimulus of exsheathirg fluid in selfcure of sheep infested with Haemonchus contortus.
Nature 183: 553-554. TERRY R. J. 1968. Applications of immunology to helminth diseases. In Immunity to Parasites (Edited by TAYLOR A. E. R.). Blackwell Scientific, Oxford. URQUHART G. M., MCINTYRE W. I., MULLIGAN W., JARRETT W. F. H. & SHARP N. C. C. 1963. Vaccination against helminth disease. International Veterinary Congress 17 (Hanover) 1: 769-774.
for Parasitology.
1975. Vol. 5. pp. 427430.
Pergamon Press. Printed in Great Britain.
FAILURE TO VACCINATE LAMBS AGAINST HAEMONCHUS CONTORTUS WITH FUNCTIONAL METABOLIC ANTIGENS IDENTIFIED BY IMMUNOELECTROPHORESIS Department
J. T. M. NEILSON of Veterinary Science, University of Florida, Gainesville, Florida 32611, U.S.A. (Received 19 August 1974)
Abstract-NEILsoN
J. T. M. 1975. Failure to vaccinate lambs against Haemonchus contortus with functional metabolic antigens identified by immunoelectrophoresis. Znfernational Journal for Pamsitology 5: 427-430. Metabolic products from Haemonchus contortus larvae cultured in vitro from the infective third to fourth stage were collected and concentrated. Chromatographic and immunoelectrophoretic analyses were made to study the numbers and activity of antigens in the metabolic products derived from the in vitro cultured larvae. Three-month-old lambs were given a series of injections of metabolic antigens with and without adjuvant at dose rates of 0.05, 0.5 and 5.0 mg antigen protein per injection. These animals and saline injected controls were each challenged with 3000 H. contortus infective larvae after the last antigen injection and killed 35 days later. No difference was seen in the faecal worm egg counts or the differential worm counts among the vaccinated and control animals. The antigen preparation of worm metabolic products conferred no resistance to challenge infection with the parasite. INDEX KEY WORDS: Huemonchus contorfus; lambs; metabolic antigens; immunoelectrophoresis; vaccination.
INTRODUCTION EFFORTS to vaccinate
animals with somatic extracts of nematodes have been largely unsuccessful. The antigens which induce protective immunity are probably produced only in small amounts by the living parasite and appear to be primarily associated with developing larval stages rather than adult worms (Silverman, 1970). If the host is inoculated with antigens prepared from dead worms, the functional antigens are not present in sufficient quantity, are destroyed during the extraction procedure or are concealed by the large amount of nonfunctional foreign material made available to the host’s reticuloendothelial system (Sinclair, 1970). Christie & Brambell (1966) induced a strong resistance to challenge infection with H. contortus in 2-3-month-old lambs by first exposing them to several doses of H. contortus infective larvae followed by anthelmintic treatment which destroyed the larvae before they reached the adult stage. Silverman (1965) obtained protection against H. contortus in 4-6-month-old lambs with two vaccines. One vaccine was prepared from third and fourth stage larvae somatic and metabolic antigens and the other from fourth stage larvae somatic and metabolic antigens which, when administered to lambs, gave 50 and 64 per cent reduction in challenge worm burdens respectively. In the present work, an attempt was made to
identify functional antigens in the metabolic products released by third stage H. contortus larvae following exsheathment and ecdysis to the fourth stage. Functional antigens were defined by the criteria proposed by Dineen (1963). The concentrated metabolic antigens derived from in vitro culture of H. contortus larvae were injected into lambs in an attempt to stimulate resistance to challenge with this parasite. MATERIALS
AND METHODS
H. contortus infective stage larvae were cultured to the fourth stage in vitro according to the method of Neilson (1969). Under these conditions over 90 per cent of the larvae exsheath within 48 h in culture and 70 per cent of these complete the moult to the fourth stage after 72 h. The culture medium does not contain macromolecules. This facilitates the identification and isolation of macromolecular excretions and secretions or metabolic productsliberated into the culture medium by the developing parasite (Neilson, 1969). In addition, Neilson (1969) described the fractionation of the parasite metabolic products by Sephadex gel filtration and polyacrylamide gel electrophoresis. Antisera to the unfractionated metabolic products were produced in rabbits and sheep. Three rabbits, two months of age at the start of the experiments were each injected intramuscularly with 1.0 ml metabolic products (0.5 mg protein) plus 1.0 ml Freund’s complete adjuvant. Thirty days later, 1.0 ml of metabolic products plus 427
428
J. T. M.
1.0 ml adjuvant were injected into the footpads. Fourteen days later, this was followed by a series of three intradermal injections of metabolic products (1.0 ml/injection) at weekly intervals. The rabbits were bled 1 week after the final injection and the serum from each animal pooled. Three sheep, 3 months of age at the start of the experiments and reared parasite free, were each given two intramuscular injections of 10 ml metabolic products (5.0 mg protein) plus 10 ml Freund’s complete adjuvant 30 days apart. After 14 days, this was followed by a series of three intradermal injections of metabolic products (10 ml/injection) at weekly intervals. The sheep were bled one week after the final injection and the serum from each animal pooled. Immunoelectrophoresis of the metabolic products and the Sephadex fractions thereof was done on the immunoelectrophoretic equipment supplied by the Gelman Instrument Co.* and the methods used in the entire procedure were those described by the manufacturer. Standard die 71649 (Gehnan Instrument Co.) was used to punch the desired pattern on each agar coated slide. This pattern gave one central well (1 mm dia.) and two parallel troughs (1 mm broad) on each side of the well, which allowed 3.6 mm diffusion distance between the well and each trough. The metabolic products preparation or the Sephadex samples thereof were placed in the central well, electrophoresed, then one trough was charged with sheep anti metabolic products serum while the other trough received rabbit anti metabolic products serum. In this way a direct comparison could be made of the precipitating antibodies in rabbit versus sheep antisera to the parasite metabolic products and Sephadex fractions thereof. Twenty-three lambs approximately 3 months of age, reared under worm free conditions, were distributed into 5 groups. Groups 1, 2, 3 and 4 contained 5 animals per group while group 5 contained 3 animals. Each lamb in groups 1,2 and 3 was injected on each occasion with 0.1, 1.0 and 10 ml (equivalent to 0.05,0.5 and 5.0 mg protein) H. contortus metabolic antigen respectively, according to the following schedule: Two injections intramuscularly with the appropriate volume of Freund’s complete adjuvant on day 1 and day 28 followed by a series of intradermal injections of antigen preparation on days 42, 49, 56 and 63. Group 4 animals were similarly injected with saline, 10 ml with or without adjuvant being administered on each occasion. The lambs in Group 5 were untreated. One week after the final injection, on day 70 of the experiment, all lambs were each infected orally with 3000 H. contortus infective larvae. Faecal worm egg counts were performed by the McMaster technique on 2 g faecal samples. The first faecal sample was collected on day 13 postinfection then at 3 or 4 day intervals thereafter. All animals were killed on day 35 after infection and the total number of adult worms and larval stages in each animal counted. The procedure for the recovery and counting of worms has been described (Dineen, Donald, Wagland & Offner, 1965). RESULTS Tmmunoelectrophoresis * Gelman Instrument Michigan 48106.
of the metabolic
products
Co., P.O. Box 1448, Ann Arbor,
NEILSON
I.J.P. VOL.
from in vitro cultured H. contortus larvae and logous antisera prepared in sheep and rabbits 4 and 7 precipitin arcs respectively (Fig. 1). additional precipitating antibodies had been RaES
m--
-
ES
-0
_‘cl,
SaES
---
ES8
0
SaES
-
RaES
-
ES9 SaES
-
-
ES6
RaES
homoyielded Thus 3 stimu-
0
SaES
RaES
5. 1975
x 0
-
FIG. 1. The major precipitin bands obtained following immunoelectrophoresis of unfractionated metabolic antigens (ES), fraction 2 from Sephadex GlOO column (ES6), and fractions 1 and 2 from Sephadex G200 column (ES8 and ES9 respectively) against rabbit and sheep antisera to ES (RaES and SaES respectively). lated in rabbits. One of the responsible antigens had an anodal distribution and the other two had a cathodal distribution. The antigen with anodal electrophoretic mobility was present in fraction 2 from Sephadex G-100 gel filtration while the two antigens with cathodal electrophoretic mobility were located in fractions 1 and 2 from Sephadex G-200 gel filtration. The elution profiles with fraction numbers obtained following gel filtration of the H. contortus metabolic products on Sephadex G50, GlOO and G200 have been reported (Neilson, 1969). The injection of sheep with metabolic antigens derived from in vitro cultures of H. contortus in which larvae developed from the infective third to the fourth stage, failed to confer resistance to challenge with the parasite. No difference in the pattern of faecal egg counts or the number of adult worms found at necropsy was seen among the sheep injected with any of the three doses of the metabolic rntigen preparation, the saline injected controls or sheep given no injections of any kind. The worm burdens are shown in Table 1. Furthermore, no immature or inhibited larval forms of the parasite were noted in the enzymic digests of the abomasums sf any of the sheep.
I.J.P.
VOL.
5.
1975
TABLE
~-MEAN
WORMS
RECOVERED
Antigens of H. contortus f
S.E. ON DAY
OF 3000
Group No.* 1
2 3 4 5
ADULT
Haemonchus contortus
3.5FOLLOWING
LARVAE
PER
AN INFECTION
LAMB
Worm burden 1600 f 190 2140 f 225 1860 * 325 1133 f 88 1600 & 272
*Groups 1, 2 and 3 injected 6 times (see text for injection schedule) with 0.1, 1.0 and 10 ml (equivalent to 0.05, 0.5 and 5.0 mg antigen protein) respectively. Group 4 was saline injected controls and Group 5 received no injections. DISCUSSION Soulsby, Somerville & Stewart (1959), on the basis of serological evidence, proposed that resistance to H. contortus infection in sheep was probably stimulated by antigens released during exsheathment, the terminal part of the second ecdysis and during the third ecdysis. These and other studies have indicated that probably the most logical and rewarding approach to the development of a helminth vaccine would be to cultivate the worm in vitro through those stages which are known to be potentially most immunogenic and to collect the metabolic products released which supposedly contain the functional antigens (Terry, 1968). This rationale was followed in the present study. H. contortus infective stage larvae were allowed to exsheath, then moult to the fourth stage in vitro in a balanced salt solution. The macromolecular substances released by the larvae were isolated and concentrated by methods which would minimize their denaturation (Neilson, 1969). Dineen (1963) proposed that, by a comparison of the serologic responses evoked by parasite antigens given parenterally to the parasite’s natural host and to unnatural hosts, it would be possible to discriminate between functional and casual antigens. Functional antigens are those which stimulate immune responses which adversely affect the biotic potential of the parasite and as such would be suitable components of a vaccine against that parasite. The comparative serologic responses of sheep and rabbits to parenterally injected somatic antigens of H. contortus larvae and adult worms indicated that both contained common functional antigens (Dineen, 1963). It has been shown by others that a vaccine prepared from somatic and metabolic antigens derived from third and fourth stage H. contortus larvae offered a measure of protection in 4-6-monthold lambs against a challenge infection with this parasite (Silverman, 1965). It is possible that this successful vaccine contained the functional antigens described by Dineen (1963) as well as the functional antigens in the excretions and secretions described in the present paper.
429
No protection was obtained in the present work with lambs injected with three dose levels of metabolic products, despite the fact that this preparation contained serologically indicated functional antigens. These findings would suggest that a combination of somatic and metabolic antigens from H. contortus larvae are essential components for a successful non-living vaccine against this parasite. As stated earlier, however, the metabolic products used here, contained, in addition to the serologically identified functional antigens, several nonfunctional antigens. It is possible that the presence of these nonfunctional antigens in the vaccine blocked or otherwise interfered with the induction of protective responses. In addition, earlier electrophoretic studies of the metabolic products (Neilson, 1969) indicate that several proteins in the preparation are not precipitinogens. Purification of those precipitinogens which are functional antigens should be possible by a two step procedure. First, the metabolic products are absorbed with its homologous sheep antiserum so removing the antigens which stimulate common precipitating antibodies in sheep and rabbits. Second, the antigens which stimulate the additional precipitating antibodies in rabbits can be specifically absorbed from the metabolic products by immunosorption on immunosorbent columns prepared with the homologous rabbits antiserum. Further protection studies against H. contortus in sheep should be undertaken using as vaccines various combinations of functional metabolic antigens and functional somatic antigens identified and purified in the manner described above. Three-month-old lambs were used in the present experiment whereas Silverman (1965) in a similar experiment used 4-6-month-old lambs. Urquhart, McIntyre, Mulligan, Jarrett & Sharp (1963) successfully obtained protection to H. contortus in sheep, 6 months or older, following vaccination with irradiated larvae. No such protection was found in similarly vaccinated lambs less than 6 months of age and these workers concluded that these young lambs may not be sufficiently immunologically mature. However, Christie & Bambell (1966) immunized 2-3-month-old lambs by giving several large doses of H. contortus infective larvae followed by two anthelmintic treatments. This pattern of infection followed by anthelmintic treatment was repeated then the lambs and previously uninfected controls were each given a massive challenge. Good protection against the challenge was obtained in the previously exposed lambs and it is interesting to note that the protection obtained in these 2-3-month-old lambs was greater than that obtained in a similar experiment with 8-month-old lambs (Christie, Brambell & Charleston, 1964). Hence no evidence of immunological immaturity in lambs less than 6 months of age was noted in these studies.
430
J. T. M. NEILSON
Acknowledgement-Part
of this work was done at the McMaster Laboratory, CSIRO, Sydney, Australia. This investigation was supported in part by Research Grant GB-18991 from the National Science Foundation. Florida Agricultural Experimental Stations Journal Series No. 5520.
I.J.P. VOL. 5. 1975
NEIUON J. T. M. 1969. Gel filtration and disc electrophoresis of a somatic extract and excretions and secretions of Haemonchus contortus larvae. Experimental Parasitology 25: 131-141. SILVERMAN P. H. 1965. Some immunologic aspects of parasitic helminth infections. American Zoologist 5:
153-163. REFERENCES CHRISTIE M. G. & BRAMBELL M. R. 1966. Acquired resistance to Haemonchus contortus in young lambs.
Journal of Comparative Pathology 76: 207-216. CHRISTIE M. G., BRAMBELL M. R. & CHARLESTON W. A. G. 1964. Resistance to a challenge infection with Haemonchus contortus conferred by previous experience to immature stages only. Journal of Com-
parative Pathology 74: 427434. DINEEN J. K. 1963. Antigenic relationship between host and parasite. Nature 197: 471472. DINEEN J. K., DONALD A. D., WAGLAND B. M. & OFFNER J. 1965. The dynamics of the host-parasite relationship. III. The response of sheep to primary infection with Haemonchus contortus. Parasitology 55:
515-525.
SILVERMAN P. H. 1970. Vaccination: Progress and problems. In Immunitv to Parasitic Animals (Edited bv JACKSON G. J.). Appleton-Century-Crofts, hew York. SINCLAIR I. J. 1970. The relationship between circulaticg antibodies and immunity to helminthic infections.
Advances in Parasitology 8: 97-138. SOULSBY E. J. L., SOMERVILLE R. I. & STEWART D. F. 1969. Antigenic stimulus of exsheathirg fluid in selfcure of sheep infested with Haemonchus contortus.
Nature 183: 553-554. TERRY R. J. 1968. Applications of immunology to helminth diseases. In Immunity to Parasites (Edited by TAYLOR A. E. R.). Blackwell Scientific, Oxford. URQUHART G. M., MCINTYRE W. I., MULLIGAN W., JARRETT W. F. H. & SHARP N. C. C. 1963. Vaccination against helminth disease. International Veterinary Congress 17 (Hanover) 1: 769-774.
