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Infection with Haemonchus contortus in sheep and the role of adaptive immunity in selection of the parasite
InternotionolJour~lfor
Porasifology Vol. 18, No. 8, pp. 1071-1075.1988.
Printed in Grear Britain.
01988
0020-7519/88 $3.00 + 0.00 Pergam0n Press plc Austmlian Societyfor Parosilology.
INFECTION WITH HAEMONCHUS CONTORTUS IN SHEEP AND THE ROLE OF ADAPTTVE IMMUNITY IN SELECTION OF THE PARASITE D. B. ADAMS CSIRO Division
of Animal
Health,
(Received
Pastoral
Research
Laboratory,
ArmidaIe,
NSW 2350, Australia
24 February 1988; accepted 26 July 1988)
Abstract-ADAMS D. B. 1988. Infection with Haemonchus contortus in sheep and the role of adaptive immunity in selection of the parasite. International Journalfor Parasitology 18: 1071-1075. A series of infections with Haemonchus contortus in immune and non-immune sheep gave no indication that successive generations of the parasite were selected for the enhanced ability to cope with host-protective immunity. Separate subpopulations of the parasite were passaged through individual immune sheep and were compared at each generation with infections by the pooled populations in an additional panel of immune sheep. The experiment ceased when infections became too low to allow the production of sufficient infective larvae for reinfection. Results showed that H. conforms is unable to make adjustments to the immune status of a given sheep. Attention is thus diverted from the process through which host identifies self and non-self and from the histocompatibility system as the targets for possible antigen-mimicry by H. contortus. INDEX KEY WORDS: Haemonchus contortus; nematode; acquired immunity; adaptive immunity; immunosuppression;
INTRODUCTION
THERE is some evidence that the adaptations for coping with host-protective immunity which are employed by the enteric nematode parasite, Huemon&us contortus, operate as mechanisms for mitigating the intensity of responses mounted by its host, the sheep. Depressed reactivity to antigens from H. contortus occurs in populations of lymphocytes obtained from sheep at times corresponding to the absence of protective immunity; for example, from immune ewes around the time of parturition (Chen & Soulsby, 1976) or from animals given a first infection (Adams, 1978). Other experimental results indicate that suppressor cell activity which dampens protective immunity is stimulated by first contact with parasite antigens (Adams & Davies, 1982; Shubber, Lloyd & Soulsby, 1984). Finally, functional immunological tolerance or unresponsiveness has been demonstrated when sheep are first exposed to the parasite (Adams, 1983). The demonstration of a given mechanism for coping with host-protective immunity does not, however, exclude the possibility that additional mechanisms may operate. Accordingly, the present purpose was to investigate whether other adaptations by H. contortus either for avoiding recognition by the immune system of the sheep or for surviving immunological attack could be discerned. Under a scheme of the generalized immune response described by Miller (1968), these possibilities could occur, in turn, within the afferent limb of the immune response where antigen recognition takes place or within the efferent
parasitic; sheep; ruminant; selection; host-specificity;
immune response; immune-evasion.
limb where effector activity is undertaken. By contrast, the dysfunctions associated with immunological unresponsiveness already put forward as mechanisms for immune-evasion by H. contortus would be placed within the central component of the immune response. The method adopted for discerningthe possibilities mentioned is serial passage of the parasite through immune or non-immune sheep and monitoring for any accommodation to host-protective immunity in each successive generation. An important component of this approach is the passage of separate subpopulations or ‘infrapopulations’ (Margolis, Esch, Holmes, Kuris & Schad, 1982) of the parasite through individual immune sheep. The aim is to determine whether adjustments can be made by populations of H. contortus either to the individual character of a given immune sheep or to the protective immune response of sheep in general. MATERIALS AND METHODS Sheep. Finewool Merino wethers raised at pasture under conditions designed to exclude infection by enteric nematodes (Le Jambre, 198 1) were employed. Such animals are designated worm free until deliberate experimental infection. During experiments, sheep were housed indoors on hardwood slatted floors and were fed a complete pelleted ration composed of lucerne hay, wheat, Pollard and bran. Because sheep maintained under these conditions for long periods are prone to urolithiasis (with ammonium phosphate the major component of calculi), the ration was supplemented with 1% w/w rock salt. Sheep were also injected parenterally every 2 months with a preparation of
1071
1072
D. B. ADAMS
vitamins A, D and E (Janajec,
Roche-Maag Australia Bankstown, NSW). Parasites. Methods for obtaining infective larvae of H. contortus for giving infections to sheep by intraruminal injection and for performing faecal egg counts have been described previously (Adams & Beh, 198 1). The strain of H. contortus used is known as ‘Chiswick’ wild-type and was described by Le Jambre, Southcott & Dash (1976). Oxfendazole (Systemex, Cooper Australia Ltd) was used at 9 mg kg-’ liveweight (that is, at twice the recommended dose) to terminate infections in sheep. Experimental design. In Experiment 1, two randomlyallocated groups of eight wethers, 19.9 + 2.4 kg liveweight (mean + SD.) and 4-6 months old at the outset were given a first infection with 5000 larvae which was allowed to persist for 19 weeks until treatment by anthehnintics. A second infection with 5000 larvae was given 1 week later and was terminated with anthehnintic after a further 10 weeks. After a lapse of 1 week, a third infection with 5000 larvae was given and the experiment finished 9 weeks later. Faecal egg counts were made at weekly intervals during the three infections and were commenced after a prepatent period of 21 days. Larvae for second and third infections were obtained from cultures of faeces collected from each sheep in the first group during the last 2 weeks of the preceding infection. Each animal in the first group was reinfected with larvae derived from the particular populations of adult worms they themselves harboured in the previous infection. These sheep are designated ‘infrapopulation-reinfected’. Sheep in the second group were reinfected with larvae from a pool derived in equal numbers from each donor sheep in the first group. These sheep are designed ‘suprapopulationreinfected’. In Experiment 2, 12 wethers, 27.6 f 5.3 kg liveweight (mean + SD.) and 8-10 months old at the outset, were divided randomly into two groups of six. These animals were given two immunizing infections with 10,000 H. contortus larvae while still at pasture. The first lasted 10 weeks before termination with anthelmintic. The second was given 1 week later and lasted 6 weeks before sheep were housed, treated with anthehnintic and randomly allocated into two groups. A preliminary challenge infection was then given with 10,000 larvae. It lasted 7 weeks before treatment with anthelmintic and was assessed after patency by four faecal egg counts made at intervals of 7 days. During this period, faecal samples for cultivation of infective larvae were collected from each of the sheep in the first group. First challenge infection was then given with larvae in doses of 20,000. As in Experiment 1, individuals in the first group received larvae derived from the particular population of adult worms they themselves harboured during preliminary reinfection (‘infrapopulation-reinfected’). Sheep in the other group received larvae from a pool derived equally Limited,
from each of the donor sheep in the first group (‘suprapopulation-reinfected’). The first challenge infection lasted 7 weeks until termination with anthelmintic. After patency, faecal egg counts were made at intervals of 7 days and faecal samples for cultivation of larvae were obtained from each sheep in the first group during this period. A second challenge infection was given a week after termination of the first test-reinfection. Again, ‘infrapopulation-reinfected’ sheep in the first group and ‘suprapopulation-reinfected sheep in the second group were given larvae derived from the first challenge infection according to the format used for giving the first challenge infection. Third, fourth and fifth challenge infections followed the same procedure except for minor variations in the duration of each and the use of doses of 40,000 infective larvae in the fifth challenge infection. For
serial passage in worm-free used for each generation.
sheep, a new naive animal was
RESULTS
Experiment
1 faecal egg counts recorded from the three sequential infections with 10,000 H. contortus infective larvae delivered to initially worm-free sheep in Experiment 1 are shown in Fig. 1. Faecal egg counts were significantly lower in the second and third infections compared with the first (PC 0.001 for each comparison; by paired sample t-tests on the cumulative faecal egg counts during the first 6 weeks after patency). These lower egg counts indicate the presence of host-protective immunity. Thus, it was only during the second infection when secondary responsiveness clearly operated that acquired immunity may have imposed any selection pressure on the parasite. It may have only been during the third infection that the consequences of this selection would have appeared. The apparently lower egg counts during the third infection in the group of sheep reinfected with the infrapopulations (that is, with the larval progeny of the worms they individually harbourecl during the second infection) compared with the sheep reinfected with the suprapopulation were not statistically significant. Unfortunately, a fourth infection was not possible because wool-biting began to occur among the sheep. The
Experiment 2 The design of Experiment 2 differed from Experiment 1 in that immunizing infections were given before sheep entered the animal house and selection of each infrapopulation against the acquired immunity of the host occurred five times. The faecal egg counts recorded in the infrapopulation and suprapopulation infected sheep during the six reinfections are shown in
Fig. 2. There was no indication from faecal egg counts of any selection by the acquired immune response. The last faecal egg count during reinfection 2 was higher in the infrapopulation-reinfected compared with the suprapopulation-reinfected group (P < 0.05 by paired sample t-test). However, this difference was not repeated in reinfection 3. The results of the comparison of the infectivity for worm-free sheep of the suprapopulation obtained after reinfection 3 in immune sheep and the starting population which had been passaged in parallel through successive naive sheep have been published elsewhere (Adams, 1986). In brief, the infections established from either population were similar in size. However, significantly more arrested forms were found in sheep given larvae from the primary infected donors compared with those given larvae from immune sheep (440 vs 125); a result which was accentuated when recipients were treated with dexamethasone (1400 vs 525).
H. contortus in sheep Primary
1073
First
infection
reinfection
Second reinfection
225r
200
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150
; 125L
125
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5 6 7 8 9 10 11 12 13 l4 15 16 17 18 Time after
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-
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150
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75 -
50-
50 -
25-
25 -
L
o-o
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infectiodweeks)
FIG. 1. Faecal egg counts in Experiment 1 in sheep given three consecutive infections with 10,000 Haemonchus contortuS larvae; with anthelmintic treatment at the end of each infection. Values are means k S.E.M. of square root-transformed egg counts: o----o, egg counts in sheep infected with the pooled suprapopulation of the parasite; O----O, egg counts in sheep infected with infrapopulations of the parasite derived from previous infections in the same individual host.
DISCUSSION The principal finding in the present study is that only six serial passages of Haemonchus contortus were possible in immune sheep before great difficulty was found in obtaining sufficient infective larvae for a further challenge infection. The lack of enhanced ability to cope with host-protective immunity in successive generations of the parasite virtually rules out the possibility of adjustments to withstand immunological affector mechanisms or that modifications made to parasite antigens, for instance through antigen mimicry (Damian, 1964) or reductions in antigenic disparity (Dineen, 1963), allow for evasion of immune recognition. As to possible hostdeterminants of antigen mimicry or antigenic disparity, infrapopulations of H. contortus did not adjust to fit the immune system of the individual sheep through which they were passaged. This finding turns attention from the structures through which mammals in general discriminate between self and non-self. In mind here is the histocompatibility complex (Sachs, 1984). The only effect of selection with host-protective
immunity in the present sfudy was a reduction in the frequency of variants of H. contortus likely to go into developmental arrest (reported elsewhere; Adams, 1986). Because parasites genetically endowed with the capacity for outliving host-protective immunity were not obvious, two genetic mechanisms can be largely discounted for H. contortus in this connection. The first concerns sexual reproduction and the assortment and re-assortment of genes from within a diverse pool. This may operate within H. contortus for the development of resistance to anthehnintics whether or not the inheritance of this trait is polygenic or due to major genes (Le Jambre, 1985). Genes for resistance to anthehnintics are likely to exist preadaptively at some unknown frequency (Le Jambre, 1978) and are only given prominence once the selection pressure of anthehnintic treatment is applied. By contrast, the present frequency in H. contortus of genes for resistance to the adaptive immunity of sheep can be postulated to result from longstanding selection by host-protective responses. If this is so, further selection by host-protective immunity may not be effective; as the present results bear out.
1074
D. B.
ADAMS
Preliminary reinfection
Reinfection
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FIG.2. Faecal egg counts in Experiment 2 in sheep given a sequence of infections with increasing numbers of Haemonchus contortus larvae; with anthehnintic-treatment at the end of each infection. Values are means f S.E.M.of square root-transformed egg counts: l -- - -0, egg counts in sheep infected with the pooled suprapopulations of the parasite; 0- - - -0, egg counts in sheep infected with infrapopulations of the parasite derived from previous infections in the same individual host.
The secQnd genetic mechanism for generating variants of H. contortus which could outlive hostprotective immunity, and which was found against by the present study, concerns those processes for gene re-arrangement and modification that operate apart from spontaneous mutation. The mechanism associated with the variable surface glycoproteins of trypanosomes (Vickerman, 1978) is a good example here because it may represent an established adaptation for the avoidance of antigen-recognition. Six serial passages of H. contortus through immune sheep in the present study should have been sufficient to demonstrate the activity of a similar established adaptation in this parasite. They did not.
Because no accommodation to adaptive immunity was observed in H. contortus in the present study, attention can be redirected from both the afferent (or recognition) and the efferent (or effector) limbs of the immune response and placed on the central phase as the likely point of impact for adaptations by which H. contortus evades host-protective immunity. In this regard, immunological unresponsiveness to the parasite has already been demonstrated in sheep in clinically relevant experimental situations (Manton, Peacock, Poynter, Silverman & Terry, 1962; Urquhart, Jarrett, Jennings, McIntyre & Mulligan, 1966; Chen & Soulsby, 1976; Adams, 1978; Adams & Davies, 1982; Shubber, Lloyd & Soulsby, 1984).
H. contortus in sheep Acknowledgements-I am grateful to Mr B. H. Anderson for expert technical assistance and to Mr B. Dennison for care of the sheep.
REFERENCES ADAMS D. B. 1978. The induction of selective immunological unresponsiveness in cells of blood and lymphoid tissue during primary infection of sheep with the abomasal nematode, Haemonchus contortus. Australian Journal of Experimental Biology and Medical Science 56: 107-l 18. ADAMS D. B. & BEH K. J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology 11: 381-386. ADAMS D. B. & DAVIES H. I. 1982. Enhanced resistance to infection with Haemonchus contortus in sheeo treated with a corticosteroid. International Journal for Parasitology 12: 523-529. ADAMS D. B. 1983. Investigation with dexamethasone of the processes which moderate immunity against the nematode, Haemonchus contortus, in sheep. Australian Journal of Experimental Biology and Medical Science 6 1: ?A%‘?
<”
.
ADAMS D. B. 1986. Developmental arrest of Haemonchus contortus in sheen treated with corticosteroid. International Journal for Parasitology 16: 659-664. CHEN P. & SOULSBY E. J. L. 1976. Haemonchus contortus infection in ewes: blastogenic responses of peripheral blood leukocytes to third stage larval antigen. International Journalfor Parasitology 6: 135-141. DAMIAN R. T. 1964. Molecular mimicry: antigen sharing by parasite and host and its consequences. The American Naturalist 98: 129-149. DINEEN J. K. 1963. Antigenic relationship between host and parasite. Nature (London) 197: 471-472. LE JAMBRE L. F., SOUTHCOTTW. H. & DASH K. M. 1976. Resistance of selected lines of Haemonchus contortus to thiabendazole morantel tartrate and levamisole. InternationalJournalfor Parasitology 6: 217-222. LE JAMBRE L. F. 1978. Anthelmintic resistance in gastro-
1075
intestinal nematodes of sheep. In: The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia (Edited by DONALD A. D., SOUTHCOTT W. H. & DINEEN J. K.), pp. 109-120. CSIRO Saleable Publications, Melbourne. LE JAMBRE L. F. 1981. Eradication of levamisole-resistant Ostertagia. Australian Veterinary Journal 51: 99-100. LE JAMBRE L. F. 1985. Genetic aspect of anthelmintic resistance in nematodes. In: Resistance in Nematodes to Anthelmintic Drugs (Edited by ANDERSON N. & WALLER P. J.), pp. 97-106. CSIRO Australia and Australian Wool Corporation, Glebe, NSW, Australia. MANTON V. J. A., PEACOCK R., POYNTER D., SILVERMAN P. H. & TERRY R. J. 1962. The influence of age on naturally acquired resistance to Haemonchus contortus in lambs. Research in VeterinaryScience 3: 308-314. MARGOLIS L., ESCH G. W., HOLMES J. C., KURIS A.M. & SCHAD G. A. 1982. The use of ecological terms in parasitology (report by an ad hoc committee of the American Society of Parasitologists). Journal of Parasitology 68: 131-133. MILLER J. F. A. P. 1968. Biology of the immune (allergic) response. In: Clinical Aspects of Immunology, Second Edition (Edited bv, GELL P. G. H. & COOMBS R. R. A.). \ pp. 289-33. Blackwell Scientific Publications, Oxford. ” SACHS D. H. 1984. The maior histocompatibility complex. In: Fundamental Immunblogy (Edited by PALL W:E.), pp. 303-346. SHUBBER A. H., LLOYD S. & SOULSBY E. J. L. 1984. Immunological unresponsiveness of lambs to infection with Haemonchus contortus. Effect of infection in the ewe on the subsequent responsiveness of lambs. Zeitschrifr fir Parasitenkunde 70: 2’19-228. UROUHART G. M.. JARRETT W. F. H.. JENNINGS F. W.. MCINTYRE W. I.‘M. & MULLIGAN W.’ 1966. Immunity to Haemonchus contortus infection: relationship between age and successful vaccination with irradiated larvae. American Journal of Veterinary Research 27: 16451648. VICKERMANK. 1978. Antigenic variation in trypanosomes. Nature (London) 273: 613-617.
Porasifology Vol. 18, No. 8, pp. 1071-1075.1988.
Printed in Grear Britain.
01988
0020-7519/88 $3.00 + 0.00 Pergam0n Press plc Austmlian Societyfor Parosilology.
INFECTION WITH HAEMONCHUS CONTORTUS IN SHEEP AND THE ROLE OF ADAPTTVE IMMUNITY IN SELECTION OF THE PARASITE D. B. ADAMS CSIRO Division
of Animal
Health,
(Received
Pastoral
Research
Laboratory,
ArmidaIe,
NSW 2350, Australia
24 February 1988; accepted 26 July 1988)
Abstract-ADAMS D. B. 1988. Infection with Haemonchus contortus in sheep and the role of adaptive immunity in selection of the parasite. International Journalfor Parasitology 18: 1071-1075. A series of infections with Haemonchus contortus in immune and non-immune sheep gave no indication that successive generations of the parasite were selected for the enhanced ability to cope with host-protective immunity. Separate subpopulations of the parasite were passaged through individual immune sheep and were compared at each generation with infections by the pooled populations in an additional panel of immune sheep. The experiment ceased when infections became too low to allow the production of sufficient infective larvae for reinfection. Results showed that H. conforms is unable to make adjustments to the immune status of a given sheep. Attention is thus diverted from the process through which host identifies self and non-self and from the histocompatibility system as the targets for possible antigen-mimicry by H. contortus. INDEX KEY WORDS: Haemonchus contortus; nematode; acquired immunity; adaptive immunity; immunosuppression;
INTRODUCTION
THERE is some evidence that the adaptations for coping with host-protective immunity which are employed by the enteric nematode parasite, Huemon&us contortus, operate as mechanisms for mitigating the intensity of responses mounted by its host, the sheep. Depressed reactivity to antigens from H. contortus occurs in populations of lymphocytes obtained from sheep at times corresponding to the absence of protective immunity; for example, from immune ewes around the time of parturition (Chen & Soulsby, 1976) or from animals given a first infection (Adams, 1978). Other experimental results indicate that suppressor cell activity which dampens protective immunity is stimulated by first contact with parasite antigens (Adams & Davies, 1982; Shubber, Lloyd & Soulsby, 1984). Finally, functional immunological tolerance or unresponsiveness has been demonstrated when sheep are first exposed to the parasite (Adams, 1983). The demonstration of a given mechanism for coping with host-protective immunity does not, however, exclude the possibility that additional mechanisms may operate. Accordingly, the present purpose was to investigate whether other adaptations by H. contortus either for avoiding recognition by the immune system of the sheep or for surviving immunological attack could be discerned. Under a scheme of the generalized immune response described by Miller (1968), these possibilities could occur, in turn, within the afferent limb of the immune response where antigen recognition takes place or within the efferent
parasitic; sheep; ruminant; selection; host-specificity;
immune response; immune-evasion.
limb where effector activity is undertaken. By contrast, the dysfunctions associated with immunological unresponsiveness already put forward as mechanisms for immune-evasion by H. contortus would be placed within the central component of the immune response. The method adopted for discerningthe possibilities mentioned is serial passage of the parasite through immune or non-immune sheep and monitoring for any accommodation to host-protective immunity in each successive generation. An important component of this approach is the passage of separate subpopulations or ‘infrapopulations’ (Margolis, Esch, Holmes, Kuris & Schad, 1982) of the parasite through individual immune sheep. The aim is to determine whether adjustments can be made by populations of H. contortus either to the individual character of a given immune sheep or to the protective immune response of sheep in general. MATERIALS AND METHODS Sheep. Finewool Merino wethers raised at pasture under conditions designed to exclude infection by enteric nematodes (Le Jambre, 198 1) were employed. Such animals are designated worm free until deliberate experimental infection. During experiments, sheep were housed indoors on hardwood slatted floors and were fed a complete pelleted ration composed of lucerne hay, wheat, Pollard and bran. Because sheep maintained under these conditions for long periods are prone to urolithiasis (with ammonium phosphate the major component of calculi), the ration was supplemented with 1% w/w rock salt. Sheep were also injected parenterally every 2 months with a preparation of
1071
1072
D. B. ADAMS
vitamins A, D and E (Janajec,
Roche-Maag Australia Bankstown, NSW). Parasites. Methods for obtaining infective larvae of H. contortus for giving infections to sheep by intraruminal injection and for performing faecal egg counts have been described previously (Adams & Beh, 198 1). The strain of H. contortus used is known as ‘Chiswick’ wild-type and was described by Le Jambre, Southcott & Dash (1976). Oxfendazole (Systemex, Cooper Australia Ltd) was used at 9 mg kg-’ liveweight (that is, at twice the recommended dose) to terminate infections in sheep. Experimental design. In Experiment 1, two randomlyallocated groups of eight wethers, 19.9 + 2.4 kg liveweight (mean + SD.) and 4-6 months old at the outset were given a first infection with 5000 larvae which was allowed to persist for 19 weeks until treatment by anthehnintics. A second infection with 5000 larvae was given 1 week later and was terminated with anthehnintic after a further 10 weeks. After a lapse of 1 week, a third infection with 5000 larvae was given and the experiment finished 9 weeks later. Faecal egg counts were made at weekly intervals during the three infections and were commenced after a prepatent period of 21 days. Larvae for second and third infections were obtained from cultures of faeces collected from each sheep in the first group during the last 2 weeks of the preceding infection. Each animal in the first group was reinfected with larvae derived from the particular populations of adult worms they themselves harboured in the previous infection. These sheep are designated ‘infrapopulation-reinfected’. Sheep in the second group were reinfected with larvae from a pool derived in equal numbers from each donor sheep in the first group. These sheep are designed ‘suprapopulationreinfected’. In Experiment 2, 12 wethers, 27.6 f 5.3 kg liveweight (mean + SD.) and 8-10 months old at the outset, were divided randomly into two groups of six. These animals were given two immunizing infections with 10,000 H. contortus larvae while still at pasture. The first lasted 10 weeks before termination with anthelmintic. The second was given 1 week later and lasted 6 weeks before sheep were housed, treated with anthehnintic and randomly allocated into two groups. A preliminary challenge infection was then given with 10,000 larvae. It lasted 7 weeks before treatment with anthelmintic and was assessed after patency by four faecal egg counts made at intervals of 7 days. During this period, faecal samples for cultivation of infective larvae were collected from each of the sheep in the first group. First challenge infection was then given with larvae in doses of 20,000. As in Experiment 1, individuals in the first group received larvae derived from the particular population of adult worms they themselves harboured during preliminary reinfection (‘infrapopulation-reinfected’). Sheep in the other group received larvae from a pool derived equally Limited,
from each of the donor sheep in the first group (‘suprapopulation-reinfected’). The first challenge infection lasted 7 weeks until termination with anthelmintic. After patency, faecal egg counts were made at intervals of 7 days and faecal samples for cultivation of larvae were obtained from each sheep in the first group during this period. A second challenge infection was given a week after termination of the first test-reinfection. Again, ‘infrapopulation-reinfected’ sheep in the first group and ‘suprapopulation-reinfected sheep in the second group were given larvae derived from the first challenge infection according to the format used for giving the first challenge infection. Third, fourth and fifth challenge infections followed the same procedure except for minor variations in the duration of each and the use of doses of 40,000 infective larvae in the fifth challenge infection. For
serial passage in worm-free used for each generation.
sheep, a new naive animal was
RESULTS
Experiment
1 faecal egg counts recorded from the three sequential infections with 10,000 H. contortus infective larvae delivered to initially worm-free sheep in Experiment 1 are shown in Fig. 1. Faecal egg counts were significantly lower in the second and third infections compared with the first (PC 0.001 for each comparison; by paired sample t-tests on the cumulative faecal egg counts during the first 6 weeks after patency). These lower egg counts indicate the presence of host-protective immunity. Thus, it was only during the second infection when secondary responsiveness clearly operated that acquired immunity may have imposed any selection pressure on the parasite. It may have only been during the third infection that the consequences of this selection would have appeared. The apparently lower egg counts during the third infection in the group of sheep reinfected with the infrapopulations (that is, with the larval progeny of the worms they individually harbourecl during the second infection) compared with the sheep reinfected with the suprapopulation were not statistically significant. Unfortunately, a fourth infection was not possible because wool-biting began to occur among the sheep. The
Experiment 2 The design of Experiment 2 differed from Experiment 1 in that immunizing infections were given before sheep entered the animal house and selection of each infrapopulation against the acquired immunity of the host occurred five times. The faecal egg counts recorded in the infrapopulation and suprapopulation infected sheep during the six reinfections are shown in
Fig. 2. There was no indication from faecal egg counts of any selection by the acquired immune response. The last faecal egg count during reinfection 2 was higher in the infrapopulation-reinfected compared with the suprapopulation-reinfected group (P < 0.05 by paired sample t-test). However, this difference was not repeated in reinfection 3. The results of the comparison of the infectivity for worm-free sheep of the suprapopulation obtained after reinfection 3 in immune sheep and the starting population which had been passaged in parallel through successive naive sheep have been published elsewhere (Adams, 1986). In brief, the infections established from either population were similar in size. However, significantly more arrested forms were found in sheep given larvae from the primary infected donors compared with those given larvae from immune sheep (440 vs 125); a result which was accentuated when recipients were treated with dexamethasone (1400 vs 525).
H. contortus in sheep Primary
1073
First
infection
reinfection
Second reinfection
225r
200
_
175-
‘; P150g
150
; 125L
125
x100
T
25-
1, I
4
I
I
I
I
I
I
I
I
I
I
1
1
I
,
5 6 7 8 9 10 11 12 13 l4 15 16 17 18 Time after
125
loo-
100 -
-
0
150
75 -
75 -
50-
50 -
25-
25 -
L
o-o
456789
L*t I
111 I
I
l
I
456789
infectiodweeks)
FIG. 1. Faecal egg counts in Experiment 1 in sheep given three consecutive infections with 10,000 Haemonchus contortuS larvae; with anthelmintic treatment at the end of each infection. Values are means k S.E.M. of square root-transformed egg counts: o----o, egg counts in sheep infected with the pooled suprapopulation of the parasite; O----O, egg counts in sheep infected with infrapopulations of the parasite derived from previous infections in the same individual host.
DISCUSSION The principal finding in the present study is that only six serial passages of Haemonchus contortus were possible in immune sheep before great difficulty was found in obtaining sufficient infective larvae for a further challenge infection. The lack of enhanced ability to cope with host-protective immunity in successive generations of the parasite virtually rules out the possibility of adjustments to withstand immunological affector mechanisms or that modifications made to parasite antigens, for instance through antigen mimicry (Damian, 1964) or reductions in antigenic disparity (Dineen, 1963), allow for evasion of immune recognition. As to possible hostdeterminants of antigen mimicry or antigenic disparity, infrapopulations of H. contortus did not adjust to fit the immune system of the individual sheep through which they were passaged. This finding turns attention from the structures through which mammals in general discriminate between self and non-self. In mind here is the histocompatibility complex (Sachs, 1984). The only effect of selection with host-protective
immunity in the present sfudy was a reduction in the frequency of variants of H. contortus likely to go into developmental arrest (reported elsewhere; Adams, 1986). Because parasites genetically endowed with the capacity for outliving host-protective immunity were not obvious, two genetic mechanisms can be largely discounted for H. contortus in this connection. The first concerns sexual reproduction and the assortment and re-assortment of genes from within a diverse pool. This may operate within H. contortus for the development of resistance to anthehnintics whether or not the inheritance of this trait is polygenic or due to major genes (Le Jambre, 1985). Genes for resistance to anthehnintics are likely to exist preadaptively at some unknown frequency (Le Jambre, 1978) and are only given prominence once the selection pressure of anthehnintic treatment is applied. By contrast, the present frequency in H. contortus of genes for resistance to the adaptive immunity of sheep can be postulated to result from longstanding selection by host-protective responses. If this is so, further selection by host-protective immunity may not be effective; as the present results bear out.
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FIG.2. Faecal egg counts in Experiment 2 in sheep given a sequence of infections with increasing numbers of Haemonchus contortus larvae; with anthehnintic-treatment at the end of each infection. Values are means f S.E.M.of square root-transformed egg counts: l -- - -0, egg counts in sheep infected with the pooled suprapopulations of the parasite; 0- - - -0, egg counts in sheep infected with infrapopulations of the parasite derived from previous infections in the same individual host.
The secQnd genetic mechanism for generating variants of H. contortus which could outlive hostprotective immunity, and which was found against by the present study, concerns those processes for gene re-arrangement and modification that operate apart from spontaneous mutation. The mechanism associated with the variable surface glycoproteins of trypanosomes (Vickerman, 1978) is a good example here because it may represent an established adaptation for the avoidance of antigen-recognition. Six serial passages of H. contortus through immune sheep in the present study should have been sufficient to demonstrate the activity of a similar established adaptation in this parasite. They did not.
Because no accommodation to adaptive immunity was observed in H. contortus in the present study, attention can be redirected from both the afferent (or recognition) and the efferent (or effector) limbs of the immune response and placed on the central phase as the likely point of impact for adaptations by which H. contortus evades host-protective immunity. In this regard, immunological unresponsiveness to the parasite has already been demonstrated in sheep in clinically relevant experimental situations (Manton, Peacock, Poynter, Silverman & Terry, 1962; Urquhart, Jarrett, Jennings, McIntyre & Mulligan, 1966; Chen & Soulsby, 1976; Adams, 1978; Adams & Davies, 1982; Shubber, Lloyd & Soulsby, 1984).
H. contortus in sheep Acknowledgements-I am grateful to Mr B. H. Anderson for expert technical assistance and to Mr B. Dennison for care of the sheep.
REFERENCES ADAMS D. B. 1978. The induction of selective immunological unresponsiveness in cells of blood and lymphoid tissue during primary infection of sheep with the abomasal nematode, Haemonchus contortus. Australian Journal of Experimental Biology and Medical Science 56: 107-l 18. ADAMS D. B. & BEH K. J. 1981. Immunity acquired by sheep from an experimental infection with Haemonchus contortus. International Journal for Parasitology 11: 381-386. ADAMS D. B. & DAVIES H. I. 1982. Enhanced resistance to infection with Haemonchus contortus in sheeo treated with a corticosteroid. International Journal for Parasitology 12: 523-529. ADAMS D. B. 1983. Investigation with dexamethasone of the processes which moderate immunity against the nematode, Haemonchus contortus, in sheep. Australian Journal of Experimental Biology and Medical Science 6 1: ?A%‘?
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ADAMS D. B. 1986. Developmental arrest of Haemonchus contortus in sheen treated with corticosteroid. International Journal for Parasitology 16: 659-664. CHEN P. & SOULSBY E. J. L. 1976. Haemonchus contortus infection in ewes: blastogenic responses of peripheral blood leukocytes to third stage larval antigen. International Journalfor Parasitology 6: 135-141. DAMIAN R. T. 1964. Molecular mimicry: antigen sharing by parasite and host and its consequences. The American Naturalist 98: 129-149. DINEEN J. K. 1963. Antigenic relationship between host and parasite. Nature (London) 197: 471-472. LE JAMBRE L. F., SOUTHCOTTW. H. & DASH K. M. 1976. Resistance of selected lines of Haemonchus contortus to thiabendazole morantel tartrate and levamisole. InternationalJournalfor Parasitology 6: 217-222. LE JAMBRE L. F. 1978. Anthelmintic resistance in gastro-
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intestinal nematodes of sheep. In: The Epidemiology and Control of Gastrointestinal Parasites of Sheep in Australia (Edited by DONALD A. D., SOUTHCOTT W. H. & DINEEN J. K.), pp. 109-120. CSIRO Saleable Publications, Melbourne. LE JAMBRE L. F. 1981. Eradication of levamisole-resistant Ostertagia. Australian Veterinary Journal 51: 99-100. LE JAMBRE L. F. 1985. Genetic aspect of anthelmintic resistance in nematodes. In: Resistance in Nematodes to Anthelmintic Drugs (Edited by ANDERSON N. & WALLER P. J.), pp. 97-106. CSIRO Australia and Australian Wool Corporation, Glebe, NSW, Australia. MANTON V. J. A., PEACOCK R., POYNTER D., SILVERMAN P. H. & TERRY R. J. 1962. The influence of age on naturally acquired resistance to Haemonchus contortus in lambs. Research in VeterinaryScience 3: 308-314. MARGOLIS L., ESCH G. W., HOLMES J. C., KURIS A.M. & SCHAD G. A. 1982. The use of ecological terms in parasitology (report by an ad hoc committee of the American Society of Parasitologists). Journal of Parasitology 68: 131-133. MILLER J. F. A. P. 1968. Biology of the immune (allergic) response. In: Clinical Aspects of Immunology, Second Edition (Edited bv, GELL P. G. H. & COOMBS R. R. A.). \ pp. 289-33. Blackwell Scientific Publications, Oxford. ” SACHS D. H. 1984. The maior histocompatibility complex. In: Fundamental Immunblogy (Edited by PALL W:E.), pp. 303-346. SHUBBER A. H., LLOYD S. & SOULSBY E. J. L. 1984. Immunological unresponsiveness of lambs to infection with Haemonchus contortus. Effect of infection in the ewe on the subsequent responsiveness of lambs. Zeitschrifr fir Parasitenkunde 70: 2’19-228. UROUHART G. M.. JARRETT W. F. H.. JENNINGS F. W.. MCINTYRE W. I.‘M. & MULLIGAN W.’ 1966. Immunity to Haemonchus contortus infection: relationship between age and successful vaccination with irradiated larvae. American Journal of Veterinary Research 27: 16451648. VICKERMANK. 1978. Antigenic variation in trypanosomes. Nature (London) 273: 613-617.