SOME IMMUNOLOGIC ASPECTS OF PARASITIC HELMINTH INFECTIONS

Transcription

1 AM. ZOOLOGIST, 5: (1965). SOME IMMUNOLOGIC ASPECTS OF PARASITIC HELMINTH INFECTIONS PAUL H. SILVERMAN Dept. of Zoology, University of Illinois, Urbana SYNOPSIS. Studies on the host-parasite relationship of the sheep stomach worm, Haemonchus contortus suggest that important antigens which are involved in the stimulation of immunologic resistance are released during growth and development of the parasite as it penetrates, molts, and matures in the host. These antigens seem to be released and made available to the host for only short periods of time and in some instances may be present for only a few minutes. Because nematodes do not undergo multiplication in host tissues and because the rate of acquisition of parasites and their subsequent development in the host is irregular, it is difficult regularly to elicit good levels of immunologic resistance by infection procedures which utilize normal or attenuated infective larvae. The recent development of techniques for in vitro maintenance and cultivation of various parasitic stages of the parasite provide a means for collecting antigens elaborated during growth and ecdysis. Results with these materials give hope that it might be feasible to induce an immune resistance response by vaccination which in some cases may be better than that resulting flora natural infection. INTRODUCTION In the medical sense, parasitic diseases are usually associated with tropical countries although parasitism is far more widespread in the temperate zone than is generally realized. In the veterinary world the problem is geographically more widespread. Helminth parasites are of serious and increasing concern to the livestock industry. With the rapid increase in human populations and living standards there has been a demand for increased food production. Grassland research workers have improved pastures so that today an acre supports five to ten times as many livestock as it did 3 years ago. The resultant increase in the density of grazing animals has created some new problems and intensified some old ones, particularly the problem of parasite infestation. An estimate of the extent of the parasitic helminth problem has been provided by a survey of the United States Department of Agriculture and is published in this symposium in the paper by Dr. K. C. Kates. Paradoxically, although the history of parasitology is as old as, if not older than, that of its sister disciplines of bacteriology and virology, it has lagged behind in the study of immunological aspects. Because of the animals' size, descriptive studies and (153) experimental demonstrations of many helminth life cycles were published before the 18th and early half of the 19th centuries, and the associations of helminths with specific diseases were soon determined. Similar studies with bacteria required the development of suitable microscopes which were not readily available until the middle of the 19th century. Nevertheless, within a short time of the demonstration of the association of bacteria with disease, Pasteur, Koch, Cohn, Loffler, and others had cultured many pathogens in vitro and had developed highly effective vaccines against anthrax, diphtheria, tetanus, and chicken cholera. Developments in the field of virology, since the demonstration of these "filterable agents" by Iwanovski in 1892, have been even more dramatic. Although the technique of culturing viruses in vitro required the development of tissue culture procedures that were not established until recent times, the principle of vaccination was established for smallpox by Jenner in 1778 and for rabies by Pasteur in In contrast, although Japanese workers had demonstrated experimentally induced immunity to helminth reinfection in the early 19's, it was not until the reports by Stoll (1929), Miller (1931), and others (see

2 154 PAUL H. SILVERMAN 1? IG. 1. Two stages in the exsheathing process of Haemonchus contortus infective larvae. Note the cuticle undergoing splitting and the separation of the anterior cap freeing the larva. The process is the result of the excretion of exsheathing fluid from pores which are visible at the anterior end. Heyneman, 1963) that the idea was accepted that metazoan parasites stimulated an immune resistance response. The first effective field vaccine for use against helminths was introduced by Jarrett and his coworkers (1959) only a few short years ago. This live vaccine depends upon the oral administration of larvae which have been sufficiently attenuated by X-irradiation to be capable of inducing resistance without producing significant disease effects (Poynter et al., 196). This type of approach has obvious limitations, and the ultimate aim must be the preparation of sterile injectable dead vaccines. It is now clear that the fundamentals of immunology apply equally to microbial and metazoan parasitic infections; however, there are important qualitative and quantitative differences in the nature of the hostparasite relationship which affect the degree and character of the immune response. Similarly the in vitro cultivation of helminths has lagged behind the achievements of the microbiologies. Onl\ since the 195's have any significant developments in maintaining and inducing development of parasitic helminths in vitro been recorded (see reviews by Silverman 1963, 1965). No metazoan parasite of warm blooded animals has yet been maintained in vitro through successive generations. It is the purpose of this paper to consider some of the factors involved in the stimulation of the resistance-immune response to helminth infections. In order to do this it will be necessary to consider in detail the life cycle of a helminth parasite. The species to be discussed is Haemonchus contortus, the large stomach worm of sheep and cattle. Although H. contortus was chosen as a model, similar studies on Trichinella (Larsh, 1963), Ascaris (Soulsby, 1957) Nematodirus (Brunsdon, 1963) and other nematode, cestode, and trematode infections have yielded similar results, and could equally have been used to illustrate the principles outlined here. THE PARASITIC LIFE CYCLE OF Haemonchus contortus Injection process The sheep first encounters H. contortus when it ingests the infective larva from the pasture. The ensheathed third stage larva enters the rumen where it is stimulated to undergo an exsheathment process during which it elaborates a fluid which has been termed exsheathing fluid (Figs. 1 and 2 and see Silverman, 1963; Silverman and Podger, 1964). This fluid is thought to be the first antigenic stimulus presented by the parasite to its host. After exsheathment, the parasitic third stage larva passes through the reticulum and omasum and enters the gastric stomach, (the abomasum) where it finds its way into the crypts of the gastric mucosa below the mucus layer covering the mucosa (Figs. 3 and 4). The paramucosal lumen The space immediately below the mucus and the crypts contained within the villi has been termed the "paramucosal" lumen (Read, 195). The evidence indicates that

3 IMMUNOLOGY OF HELMINTH INFECTIONS 155 to be the source of the exsheathing fluid (Fig. 7). In another 48 to 96 hours the fourth stage larva, still living within the paramucosal FIG. 2. Another type o exsheathment encountered with trichostrongylid infective larvae. Splitting along the lateral line and partial formation of the cap still attached to the cast off sheath is frequently observed. the physico-chemical conditions in the paramucosal lumen are like those of the intercellular spaces of the host. This physiological stratum is a site of considerable metabolic activity. Absorption, secretion, and synthesis of nutrients and growth factors occur here. All the normal constituents of host interstitial fluids are found in the paramucosal lumen including serum globulins and other proteins. The histotropic stages By the 48th hour after ingestion, fourth stage H. contortus larvae may be found in the paramucosal lumen, having undergone their first parasitic molt (Figs. 5, 6). Evidence indicates that during the first parasitic ecdysis more exsheathing fluid as well as other metabolites are secreted (Silverman, 1963). The process of exsheathment has been studied in some detail by Sommerville (1957), and Rogers and Sommerville (1963), and it has been demonstrated to be an endocrine mechanism. Ligature experiments suggest that certain cells associated with the base of the larval esophagus appear FIG. 3. Histological cross section through a parasitic third stage H. contortus larva in situ between the mucosa and mucus layer of the gastric stomach of a sheep. FIG. 4. A parasitic third stage larva of H. contortus between the villi of the gastric mucosa of a sheep.

4 1S6 PAUL H. SILVERMAN that the nematode enters the tissues and begins to produce a pathological effect (Fig. 8). The young adults suck blood and produce ulcerations which may lead to severe anemia and even death in a susceptible sheep or lamb. The drop in haematocrit, or packed-cell volume, may be quite dramatic and death can be caused within 3-4 weeks after ingestion, if the larvae taken in have been of a sufficient number (Fig. 9). FIG. 5. A fourth stage H. contortus larva in the paramucosal lumen between the villi of the gastric mucosa of a sheep. lumen, undergoes the second and last parasitic molt becoming a fifth stage or young adult. It is after this second parasitic molt FIG. 7. Enlargement of the anterior portion of a fourth stage larva of H. contortus. The buccal capsule is formed and the granular cells that are thought to be associated with the production of exsheathing fluid can be seen located posterior to the nerve ring. FIG. 6. Fourth stage H. contortus larva in the paramucosal lumen between the mucus and mucosa of the gastric stomach of a sheep. Note the formation of the buccal capsule which characterizes the fourth larval stage. EXPERIMENTAL STUDIES ON HOST IMMUNITY On the basis of Stoll's (1929) report that sheep become resistant to H. contortus after repeated exposures to infection, an experiment was designed to investigate which histotropic stages were affected by the resistance response, and to determine the source of antigen associated with this immune mechanism (Silverman and Patterson, 196). Three groups of sheep were used: (1)

5 IMMUNOLOGY OF HELMINTH INFECTIONS 157 for histological examination, (b) The mucosa was then scraped off into warm saline. Some of the histotropic stages were quickly collected, rewashed in warm sterile Tyrode's solution and transferred into previously prepared sera from susceptible and resistant sheep, (c) Another sample of histotropic larvae was collected and pooled with similar stages from other sheep, for use in protection tests. Such larvae were either injected directly into susceptible sheep or deepfrozen for later use. (d) The remaining mucosal scrapings were fixed in formalin for detailed morphological studies of the parasitic stages. The results of these studies are graphically summarized in Figure 1. It was found SUSCEPTIBLE LAMB FIG. 8. Fifth stage H. contortiis in situ. The first signs of tissue damage are evident. worm-free lambs, (2) worm-free sheep (one year old, and (3) resistant sheep (one year old). Animals from each group were challenged with oral doses of 5,-1, H. contortus larvae and killed at regular intervals during the second to fifteenth day after infection. After slaughter, the abomasum was quickly opened and treated in the following manner: (a) Pieces of the pyloric and cardiac portions were cut out and fixed <-> 25 a. INFECTION WITH 1, LARVAE PHENOTHIAZINE GIVEN WEEKS FIG. 9. The anemia-producing effect of Haemonchus contortus. The packed cell volume (PCV) of the infected group dropped to a level which would have been fatal had the group not been cleared of worms by treatment with the anthelminthic, phenothiazine. DAYS AFTER INFECTION RESISTANT SHEEP FIG. 1. Graphic representation of results obtained in the experiment described in the text. The development of Haemonchus contortus was more rapid in young than in older animals. In resistant sheep the stages which appeared to be affected by the resistance response were those undergoing the first parasitic ecdysis and to a lesser extent the second parasitic ecdysis. that H. contortus larvae rapidly matured in young susceptible lambs aged 4 to 6 months, and that egg-producing adult worms could be found by the 15th day after infection. In older sheep, aged 8 to 12 months, which had been reared under worm-free conditions prior to the beginning of the experiment, mature adult worms appeared in numbers on about the 2th day after infection. In resistant sheep, the life cycle of the parasite proceeds to the L 4 and to a much lesser extent to the L 5 stage, but the majority of the worms (over 8%) become inhibited at these stages until they are thrown off, usually by the tenth to fifteenth day.

6 158 PAUL H. SILVERMAN FIG. 11. Formation of precipitates in the intestines of H. contortus histotropic larvae after being kept in serum from resistant sheep. Histological examination of the mucosa and the histotropic larvae in situ revealed no evidence of unusual host tissue responses. There appeared to be an increase in the number of lymphocytes and some evidence of lymphoid hyperplasia in resistant sheep as compared with susceptible animals. It has not been possible, however, to confirm this subjective impression quantitatively. The conclusion was reached that, since at the time of their inhibited development in resistant animals the larvae were in the paramucosal lumen, the resistance mechanism was affected by humoral agencies. The studies on in vitro serum reactions to (1) exsheathed third stage larvae, (2) fourth stage larvae, (3) early fifth stage larvae, and (4) mature adult worms, indicated that the fourth and early fifth stages furnished important sources of antigen(s) and were the most susceptible to the adverse effects of serum from resistant animals. Although "nonspecific" streaming precipitates at oral and excretory openings were observed, as was a tendency of larvae to agglutinate (caused apparently by anticuticular reactions which also resulted in the adherence of debris to their sticky outer surface), these reactions did not appear to affect the survival of worms even when maintained for up to 72 hours. Streaming precipitates tended to form more in immune than in normal serum, but this reaction could not be regularly associated with the state of resistance of the host. On the other hand, precipitates which formed in the intestines of fourth and fifth stage larvae maintained in serum from resistant sheep, regularly caused the death of larvae maintained overnight at 37 C (Fig. 11 and 12). Similar reactions in serum from susceptible sheep occurred only infrequently. The critical test for assessing the significance of the "functional" antigenicity of L 4 and L 5 stages was investigated by means of protection experiments. Three groups of three sheep each were used. The groups were: (A) injected with freshly recovered larvae, (B) injected with.5% formalin- FIG. 12. Extrusion of the intestine of H. contortm histotropic lanac after being kept in serum from resistant sheep.

7 IMMUNOLOGY OF HELMINTH INFECTIONS 159 TABLE 1. Summary of protection tests in sheep with Haemonchus contortus antigens derived from histotropic stages obtained in vivo. Group Number of sheep Antigen % Protection* compared with control A BC Fresh L. & early L 5 77 Formalinized L 4 & early L B 8 None 1 (Based on mean worm burden 21 days after challenge.) Mean worm burden 1,7 9 4,6 treated larvae, and (C) untreated. Several weeks later, these animals were challenged with 5 to 7 larvae. After three weeks the animals were killed, and their gastric stomachs examined for adult H. contortus. Both of the groups of sheep which had been injected with a combination of L 4 and L 5 material showed a marked reduction in the abomasal worm burden when compared with the controls (Table 1). Similar tests which incorporated groups of sheep injected with antigen preparations from third stage larvae or adults showed little or no evidence of protection (Fig. 13). IN VITRO CULTIVATION OF HISTOTROPIC STAGES The studies described above pointed up the importance of obtaining antigens associated with the developing worm. The results added further weight to the concepts propounded by Chandler and Taliaferro that "metabolites" were important antigens required for the stimulation of the resistance-immune response (see Tromba, 1962). WORM STAGES IN VITRO SERUM REACTION IN VIVO PROTECTION THIRD ~+~ + FOURTH + + EARLY FIG. 13. Summary of experimental studies designed to determine which histotropic stages were affected by the resistance response and to determine the source of antigen associated with the mechanism of immunity to Haemonchus contorlus. The results indicated that the fourth and, to a lesser extent, the early fifth stages were involved as both a source of antigen and the site of antibody action. Progress in the development of procedures for the in vitro cultivation of parasitic nematodes outside of their hosts encouraged the hope that techniques for the collection of antigens elaborated by helminths during histotropic development would soon be available. Unfortunately, the media used for in vitro culture work consisted of complex components such as tissue homogenates, sera, and other proteinaceous materials (Weinstein and Jones, 1956; Silverman, 1959; Diamond and Douvres, 196; Leland, 1961). These media greatly complicated the task of extracting and concentrating the functional antigens elaborated by the histotropic stages. Since, however, the resistance response manifested its inhibitory action at the third (i.e., the first parasitic) ecdysis, and to a much lesser extent at the fourth ecdysis, attempts were undertaken to induce on a large scale the first parasitic ecdysis in a simplified medium as had been observed by Lapage (1935) and Stoll (194). PROTECTION TESTS WITH ANTIGENS DERIVED FROM IN VITRO CULTURES Provided that suitable preculture conditions were established, it was eventually found that many species of parasitic nematodes could be stimulated to exsheath, and could then be induced to undergo rapid and homogenous development through the first parasitic ecdysis in media consisting of balanced salt solutions supplemented with a small quantity of autolyzed liver extract (Silverman and Podger, 1962; Silverman, 1963). The antigens collected from such in vitro cultures were found to be highly effective in stimulating resistance in laboratory animals to a variety of nema-

8 16 PAUL H. SILVERMAN TABLE 2. Summary of worm burdens resulting from challenge of guinea pigs with 5, Dictyocaulus viviparus larvae following injection of L k antigen at different time intervals. (After Silverman, Poynter, and Vodger, J. Parasitol., 1962.) Group Interval between injections (days) D (single injection) E Uninjected control t Worm burdens- Individual Group count total died Group mean Died from bacterial infection prior to challenge. Protection (per cent) "t" Significance test t = 3.53 highly significant t = 3.1 significant t = 1.52 not significant t = 2.38 significant TABLE 3. Summary of worm burdens resulting from challenge of rabbits with 15, (Experiment A) and 75, (Experiment B) Strongyloides papillosus infective larvae following the intraperitoneal injection of two doses (3, LE*/'dose) of homologous L t antigen administered with an interval of 21 days. The rabbits were challenged percutaneously 1 days after the second injection and killed 1 days later for worm burden examination. (After Silverman, Poynter, and Podger, J. Parasitol., 1962.) Experiment Group treatment Injected twice with 3, LE* L, antigen Uninjected controls Injected twice with 3, LE L ( antigen Uninjected controls -Worm burdens ^ Individual Group Group Protection count total mean (per cent) ,254 5,528 1, ,134 24,681 3,44 11,344 4, ,411 22, , ,368 6,259 1, LE =z larval equivalents.

9 IMMUNOLOGY OF HELMINTH INFECTIONS 161 TABLE 4. Summary of deaths resulting from challenge of guinea pigs with 2, Trichostrongylus colubritormis larvae following immunization with an L, antigen administered at different dose levels. Two intraperitoneal injections were given at an interval of 21 days. (After Silverman, Poynter, and Podger, J. Parasitol., 1962.) Dose level administered to each Number of deaths group of five guinea pigs in resulting in each larval equivalents (LE) group 1, 5, 2,5 1, uninoculated control tode infections (Silverman, Poynter and Podger, 1962). At least two injections of antigen administered at an interval of not less than 14 days yielded good protection (see Tables 2 and 3). Adjuvants were found to have a significant effect, and lyophilization did not appear to affect the antigen adversely. Evidence of cross-reactivity which was manifested by nonspecifically induced protection was also observed with some antigens. One of the most dramatic demonstrations of protectivity was obtained with the use of Trichostrongylus colubriformis, which is normally an intestinal parasite of sheep but which also produces a fatal infection in guinea pigs. Immunization with antigens derived from in vitro cultures of T. colubriformis protected against death when as little as 16 larval equivalents of antigen were used (Table 4). It was noted that somatic antigen preparations from third stage larvae and from adult worms would in some test systems induce varying degrees of measurable resistance. When these somatic antigens were compared on an equivalent weight basis with antigens derived from histotropic stages of the homologous species, the L 4 antigens were markedly more active. When titration experiments were conducted, the L 4 antigens continued to demonstrate protective stimulating properties after somatic antigens had ceased to be active at similar dilutions. It appears that the active functional antigen(s) are not exclusively found in histotropic stages, but may exist as either precursors or in an inactive form in other helminth stages. In tests with sheep, resistance to H. contortus infection was stimulated with antigens prepared from in vitro cultures of L 4 stages (group E, Table 5). The level of resistance, which was measured by worm burden counts and anemia effects, was not raised by combining L 4 antigens with exsheathing fluid or L 3 somatic extracts (groups C and D, Table 5). DISCUSSION Our studies and those reviewed by Heyneman (1963) and reported recently by Brunsdon (1963) on the stimulation of immunologic resistance, all point to the importance of antigens which are released during growth and development, particularly as the parasites penetrate, molt, and begin to mature in the host. It should be emphasized that these important antigens seem to be released and made available to the host for only short periods of time, and in some cases may be available to the host for only minutes. The release of small quantities of antigen for brief periods of time during natural infection may be inadequate to stimulate antibody formation, but could produce a low degree of hypersensitivity. Nematodes do not undergo multiplication in host tissues, and since the rate of acquisition of helminths during natural infection tends to be an irregular process dependent upon epidemiological factors, and since the rate of development of helminth larvae is dependent upon (a) the physiologic age of the infective stage, and (b) the in situ conditions of the host, the production of a quantity of the normally transitory antigen sufficient to stimulate an immune response can be, at best, a highly irregular process. The problem of inducing resistance by natural infection, which is dependent upon the presentation to the host of an adequate quantity of the appropriate antigen as outlined above, is further affected by the reactivity of the host population. It appears that, in contrast to the selective pressures

10 162 PAUL H. SILVERMAN TABLE 5. Sheep protection test with Haemonchus contortus antigens. Each group consisted of 5 lambs 4-6 months of age. Groups A, B, C, D, E received two subcutaneous injections of the stated antigens and F received a single injection. Vaccinations were made at 21 day intervals and the challenge of 5, larvae was carried out with a single oral dose 12 days afterwards. Group A B C D E F Control Antigen Live, exsheathed L 3 larvae Dead, exsheathed L 3 larvae L 3 somatic and exsheathing fluid L 3 somatic, exsheathing fluid and L, somatic and metabolites Lj somatic and metabolites L 3 somatic, exsheathing fluid L, metabolites (single injection) Total worm burden 11,868 7,496 8,965 6,848 4,948 1,378 13,592 Mean worm burden 2,375 (116-32) 1,499 ( ) 1,646 ( ) 1,369 ( ) 989 ( ) 2,75 ( ) 2,718 (19-334) % Reduction Worm burden Egg output Anemia effects Blood values as % of values at challenge Hemoglobin RBC PCV exerted by bacterial and viral diseases, there is little evolutionary evidence of a similar pressure by parasitic organisms on host populations. As a result, the problems of establishing good levels of resistance uniformly by infection procedures using normal or attenuated infective larvae are further exacerbated. Field observations on flocks and herds, which indicate animal reinfection, are usually based on egg counts and represent the results of several mechanisms. It is apparent that it is possible for animals to be resistant to reinfection although they may harbor a population of adult worms. The adult stages are not eliminated by the immune response, although they may be partially inhibited. During stress, particularly during parturition, histotropic stages which are inhibited by an immune response may undergo rapid maturation and begin egg production. Overwintering conditions depress the level of infective larvae on pasture, and animals which are kept on a low plane of nutrition tend to suffer a sharp drop in antibody titre because of a combination of lack of antigenic stimulation and loss of condition. These animals are then susceptible to reinfection in the spring. Antigens extracted from the somatic tissues of adult worms or from the free-living stages do not provide a good source of the "functional" antigens. This lack of functional antigen would explain, at least partially, the disparities between serological and resistance data and the difficulty in effecting artificial immunity. It would appear that it should be possible artificially to induce a better immune response by vaccination with adjuvanted functional antigen than is obtained with normal infections. Although antigen preparations have proven to be highly effective in inducing resistance in laboratory animals for a number of different species of nematode parasites, they have not yet answered the full demands required for practical application to domestic animals. In tests with sheep in which animals injected with the L 4 antigens were exposed to single challenge doses, marked resistance to infection was obtained (Table 5); however, it is too early to recommend this procedure as a practical immunization scheme, although under field trickle challenge conditions the level of immunity induced with l. t antigens might

11 IMMUNOLOGY OF HELMINTH INFECTIONS 163 be adequate. The accumulated evidence indicates that the antigen is incomplete. Until recently, any prolonged maintenance in vitro of the L 4 stages of helminths and the second parasitic molt took place only in complex media. However, a medium developed by Sayre, Hansen, and Yarwood (1963) for cultivation of a free living nematode was tested for Haemonchus contortus and excellent development to the late L 4 and early L-, pre-molt stage was obtained with vigorous larvae (Hansen and Silverman, unpublished data). This medium consists of 64 chemically defined components, plus a discrete proteinaceous growth-control factor. It offers the opportunity to develop larvae in vitro in the absence of macromolecular substances through their parasitic stages, and to facilitate the harvest of elaborated antigens. It is to be hoped that the exploitation of this medium will lead to the preparation of a more complete and potent immunizing antigen. REFERENCES Brunsdon, R. V A 1 'ematodirus infestation in lambs: the importance of prepatent infestation in the stimulation of resistance. N. Z. Vet. J. 11: Campbell, C. H The antigenic role of the excretions and secretions of Trichinella spiralis in the production of immunity in mice. J. Parasitol. 41: Chandler, A. C Immunity in parasitic diseases. J. Egypt. Med. Ass. 36: Diamond, L. S., and F. W. Douvres Cultivation of parasitic stages of the swine nematodes Hyostrongylus rubidus and Oesophagostomum quadrispinulatum (O. longicaudum) free of microbial associates. J. Parasitol. 46 (Suppl.):25. Heyneman, D Host-parasite resistance patterns some implications from experimental studies with helminths. Ann. N. V. Acad. Sci. 113: Lapage, G The behaviour of sterilized exsheathed infective trichostrongylid larvae in sterile media resembling their environment in ovine hosts. J. Helminth. 13: Larsh, J. E Experimental trichiniasis. p In B. Dawes, (ed.), Advances in parasitology. Academic Press, London and New York. Leland, S. E The in vitro cultivation of the parasitic stages of Cooperia punctata, Cooperia onchophora, Ostertagia ostertagia and Ostertagia circwncincta: A preliminary report. J. Parasitol. 47 (Suppl.):21. Read, C. P The vertebrate small intestine as an environment for parasitic helminths. Rice Inst. Pamph. 37 (2): Rogers, W. P., and R. I. Sommerville The infective stage of nematode parasites and its significance in parasitism, p In B. Dawes, (ed.), Advances in parasitology. Academic Press, London and New York. Sayre, F. W., E. L. Hansen, and E. A. Yarwood, Biochemical aspects of the nutrition of Caenorhabditis briggsae. Exptl. Parasitol. 13: Silverman, P. H In vitro cultivation o the histotrophic stages of Haemonchus contorttis and Ostertagia spp. Nature 183:197. Silverman, P. H. 19G3. In vitro cultivation and serological techniques in parasitology. p In A. E. Taylor, (ed.), Techniques in parasitology, Blackwell Publications, Oxford. Silverman, P. H., and J. E. Patterson Histotrophic (parasitic) stages of Haemonchus contortus. Nature 185: Silverman, P. H., and K. R. Podger Larval antigens derived by cultivation of some parasitic nematodes in simple media. J. Parasitol. 48 (Suppl.):15. Silverman, P. H., and K. R. Podger In vitro exsheathment of some nematode infective larvae. Exptl. Parasitol. 15: Silverman, P. H., D. Poynter, and K. R. Podger Studies on larval antigens derived by cultivation of some parasitic nematodes in simple media: protection tests in laboratory animals. J. Parasitol. 48: Sommerville, R. I The exsheathing mechanism of nematode infective larvae. Exptl. Parasitol. 6:18-3. Soulsby, E. J. L Some immunological phenomena in parasitic infections. Vet. Rec. 69: Soulsby, E. J. L The nature and origin of the functional antigens in helminth infections. Ann. N. Y. Acad. Sci. 113: Stoll, N. R Studies with the strongyloid nematode, Haemonchus contortus. I. Acquired resistance of hosts under natural reinfection conditions, out-of-doors. Am. J. Hyg. 1: Stoll, N. R In vitro conditions favoring ecdysis at the end of the first parasitic stage of Haemonchus contortus (Nematoda). Growth 4: Thorson, R. E Studies on the mechanism of immunity in the rat to the nematode Nippostrongylus muris. Am. J. Hyg. 58:1-15. Tromba, F. G Immunology of nematode diseases. J. Parasitol. 48: Weinstein, P. P., and M. F. Jones The in vitro cultivation of Nippostrongylus muris to the adult stage. J. Parasitol. 48:

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