Trichinella spiralis (T. spiralis) is a unique nematode parasite that spends its larval and adult life in the same host. It may infect almost any mammal, although humans, swine and rats are medically more important hosts (1-4). Microscopic newborn larvae (NBL)are produced by the female adult worms in the small intestinal wall and they traverse the mucosal tissues in the intestine and disseminate systemically in the host, causing tissue damage and occasionally death. Acquired resistance to T spiralis infections has been recognized for more than 60 years(5). It has been demonstrated that every stage of the life cycle of T. spiralis can evoke a protective host immune response during infection(6-15)and each response is stage-specific due to the uniqueness in both the cuticular antigens and the excretion-secretion antigens of each stage. NBL have been recognized to induce a strong immune response in rodents and pigs(16-20). Although many biological properties of NBL have not been well understood, immunity has been demonstrated in vivo using immunization with NBL followed by stage-specific challenge. A great deal of in vitro work has focused attention on the role of the antibody mediated granulocytic killing of the larval worms.

The effectiveness of immunity to NBL in vivo has been questioned on the belief that it occurs too late to exert an effect during the primary infection and, in a secondary infection, worm rejection usually occurs before NBL are produced. This view may be true for some rodents capable of early strong expulsion of adult worms in a primary infection, e.g., NIH, NFS, NFR mice and most inbred rats. However, in a primary infection, the period of residence of adult worms in the intestine is variable depending on host species, e.g., the pig and some inbred mice reject adult worms quite slowly (20,21), and it also depends on the genotype of hosts within a given species (21-23) as well as variables such as infection dose (24,25). Thus, if a challenge infection is sufficiently delayed after the primary infection, adult worms will develop and persist long enough to produce NBL. These observations suggest that protective immunity to NBL cannot be discounted a priori especially as the in vivo expression of anti-NBL immunity has not been assessed experimentally during natural primary or challenge infections. The following report reviews the literature and presents recent data from this and other investigators that address these questions.

Four methods have been employed to determine NBL production or the overall success of an infection in vivo. The first approach was to determine muscle larvae burdens after transplantation of single females or paired adult worms intraintestinally in rats (33,34) or mice (35,36). Only the latter provides a true estimate in a species that mates more than once. Total production per female varied from 345(34)to 1660(36). These experiments probably provide a more accurate estimate of the reproductive potential of T. spiralis rather than actual reproduction as it might not occur in infections of anything more than a minuscule number of worms.

A variant of this approach has been to determine eventual muscle larvae burden after a given muscle larvae infection (6,22,37-41). Total muscle larvae recovery in these experiments has often been considered to represent total NBL production in vivo. However, this approach is based on the assumption that all NBL produced reach and encyst in muscle tissue. Adult female worms in the intestine are often not quantitated hence the numerical base for calculating reproduction is the dose of worms given. This is not reliable in that no infection ever successfully establishes at 100% level and, numerous factors, e.g., infection dose, fecundity of females, the rejection rate of intestinal adults, the genetic influence(21,42-45), the innate resistance and specific immunity against NBL and, successful maturation of larvae in muscle can all influence the ultimate muscle larvae burden. Indeed, studies of muscle larvae burden in different inbred mouse strains given identical infections unequivocally demonstrate the strain-to-strain variations that occur in this parameter (43-45). A different method was used by Denham and Martinez (16) who administered the drug- methyridine to eliminate adult worms in the small intestine at specified intervals after initial infection. Methyridine does not affect NBL that have been produced. By examining muscle larvae burden of mice infected for 5 to 2l days before injection of methyridine the dynamics of production could be estimated. Using this method, the peak NBL production occurred 7 days after infection.

The most complete attempt to quantitate larval production and to relate this to muscle larvae burden was made by Despommier et al.(46). These authors combined direct in vitro counts of larvae produced by female worms cultured over a 24-hr period and, multiplied this by the number of female worms present in the small intestine to derive the total daily production. Cumulative estimated larvae production was found to be within 2% of total muscle larvae burden in CFW mice. Using this method, peak larvae output occurred on day 6 post infection. The same method was used by Bell et al.(45) with C₃H, NFR and DBA/1mice. These authors found that in C₃H strain, 13% fewer larvae were produced than were recovered as muscle larvae. In the other two strains, the total muscle larvae burden was only 68% and 62% respectively, of expected, based on in vitro NBL output and total intestinal female count. Muscle larvae development from a given NBL output is thus a strain specific process in mice. Furthermore in some strains, significantly fewer muscle larvae are found than there are NBL produced.

The first quantitative analysis of NBL production in vivo was carried out by Harley and Gallicchio(30)who counted NBL in thoracic duct lymph and various other tissues and organs of rats from 4 to 15 days after infection. Their results showed that NBL were first produced on day 5 after infection and that production peaked on day 9. In their experiments, the thoracic duct was the major outlet for larvae migrating from the intestine to the blood. Our own studies (47) followed the same format as those of Harley and Gallicchio (30) except that NBL output in portal vein blood was quantitated and we used acute preparations (6-24 hours) for thoracic duct lymph collection. Under these conditions, NBL output in lymph peaked at day 7 but actual output was similar for the first three days of production (6, 7, 8) and dropped precipitously thereafter. The differences in kinetics and in total larvae output in Harley and Gallicchio’s(30)experiments and ours may reflect an immunosuppressive effect produced by chronic drainage of the thoracic duct. Other possible influences, such as the strain of rat used, were eliminated through the use of common strains.

Analysis of total recovery of NBL in the three major in vivo sites of migration, the peritoneal cavity, thoracic duct lymph and blood gave numbers of NBL that were compatible with total muscle larvae burden as defined by digestion. With an infection of 4,000 muscle larvae, 10,103 NBL were recovered from peritoneal cavity, 8,238 from thoracic duct lymph, 781,200 from portal vein. The total recovery was 799,541 and the final muscle larvae burden was 615,933+159,546. Estimation of total NBL production by the in vitro techniques of Despommier et al.(46) produced a daily larval output that was kinetically very similar to our data obtained from direct in vivo quantitation(47). The major difference is that in vivo estimates of NBL output tend to be 10-20% higher than the muscle larvae burden that is found. Part of this discrepancy is due to the ability of NBL to recirculate, the net effect of which is an over estimate of NBL production in vivo.

Dennis et al. (57) examined the question by quantitating NBL infectivity after their injection intraduodenally, intravenously (iv), or intraperitoneally (ip) into mice. Specimens of diaphragm muscle of these mice were examined for muscle larvae on day 14. They found muscle larvae in the diaphragms of iv and ip injected mice only. A similar finding in rats showed that 66% of the NBL injected iv matured to muscle larvae whereas only 2% of the larvae injected ip established in muscle. The first quantitative study of natural NBL migration was again that of Harley and Gallicchio (30) described above. These authors searched for, and found, NBL in every major organ examined, as well as in the peritoneal cavity and blood. Since about 70% of the NBL recovered were from fistulae established in the thoracic duct, it was suggested that the main pathway of NBL migration was from the small intestine to draining lymphatics and then to blood and muscles. None of the above studies examined or quantitated NBL migration in the hepatic portal vein that carries all the upper intestinal venous return to the heart.

Our studies addressed this question by obtaining both thoracic duct lymph and portal vein blood samples from individual rats using acute preparations. With this method lymph was collected for 6 hours after which time rats were anesthetized and peritoneal cavity and portal vein blood samples collected. By measuring portal vein blood flow, it was found that over 97% of NBL entered capillaries directly in the intestine(47). Once in the blood, larvae rapidly traverse capillary beds in the liver and lung and can reappear in the venous blood 8 minutes after their initial introduction into the venous circulation. This is the time required by the fastest larvae to complete one full passage; however, the majority of the larvae injected in one bolus return to the starting point some 20-30 minutes after their introduction(58). By injecting a known number of NBL into the circulation and sampling the blood at intervals thereafter, we determined that about 40% of the circulating population disappears every hour. Most of these presumably penetrate striated muscle. With this speed of migration, 75% of the initial population migrate away from the blood within the first 3 hours after their introduction. A small proportion continues to circulate and larvae may still be in non-muscle tissue 24 hours after their inoculation(58).

NBL, thus, migrate through the body quite extensively before they eventually gain access to muscle cells. Relatively few appear to enter the striated muscle on their first passage through this tissue as can be seen from the following calculations: larvae recirculate in about 20 minutes and total body striated muscle mass is around 40% for most mammals. Therefore 40% of NBL should be randomly delivered to muscle in the first passage; if they all penetrated and 40% of the remainder also penetrated during the next 20 minutes when they were delivered to muscle and other tissues, then 78% of the starting population would have been removed from the circulation within the first hour. This is not observed empirically. It would appear that a large number of NBL that are delivered to muscle either remain in capillaries and pass right through the muscle or actually penetrate into the muscle tissue and then leave it and reenter the circulation. This concept is consistent with the data of Despommier et al. (59) who found that 40% of the muscle larvae recovered after direct injection of NBL into muscle were found in sites other than where they were injected. It should be mentioned that while the above experiments have provided the first estimation of the dynamics of larval migration via blood and tissue, they are based on the injection of a single bolus of larvae. It is possible that single bolus injection may influence migratory patterns in some way. This has not yet been examined, hence studies with small numbers of radiolabelled larvae are needed to confirm these figures.

The peritoneal cavity is not a significant migratory route, less than 2% of total larvae produced during a natural infection can be recovered here and only 2% of viable NBL injected i.p. matured to muscle larvae. Since from 40-60% of NBL mature successfully after their intravenous injection (45,47) the low level of maturation indicates that NBL are unable to escape the peritoneal cavity. In fact, later in the infection, we observed a degree of larval growth in peritoneal larvae that suggested partial but fruitless development of free larvae in this site. These experiments are in close agreement with those of Dennis et al. (57) and suggest that less than 0.5% of muscle larvae burden is due to NBL that have migrated from the peritoneal cavity.

Attempts were also made to estimate the time taken by NBL to penetrate draining lymphatics or capillaries in the intestine after their birth. These experiments were based on the injection of adult worms intraluminally so that an unspecified time must be allowed for adult worm penetration into their intestinal niche. Under these conditions, NBL first appeared in lymph 50.3 ± 8.5 minutes after injection of adult worms. It is likely that less than half of this period was actually taken by NBL to migrate away from the intestinal mucosa(47,58). Many questions remain to be answered vis-à-vis the mechanisms of NBL migration through the mucosal tissues in the intestine, NBL penetration of the blood vessels, lymphatics and the striated muscle cells.

Shortly after this biochemical studies of the cuticle of T. spiralis by Philipp et al. (63) and Parkhouse et al. (64) showed qualitative variations in protein composition between muscle larvae, adults and NBL. In addition, there were quantitative changes in protein expression from day 1 to day 6 in adult worms. Recent structural and biochemical studies revealed the lipid components of muscle larvae(65) and an immunogenic protease was also identified in muscle larvae(66). With new techniques available to isolate the nurse cells(68), more findings will be made. In NBL, four distinct proteins with approximate molecular weights of 64K, 58K, 30K and 28K were found(63,64). The cuticle of T. spiralis is thus a dynamic organ, which exhibits dramatic changes at the molecular level between different stages of the life cycle and more gradual modifications during development within one stage.

Despite their mundane morphology the larvae undergo what appears to be a differentiation, if not a developmental process, which is independent of their interaction with the host. These modifications are evident in the cuticular proteins as NBL maintained in vitro age. It has been suggested by at least two groups that changes in susceptibility to immunity are associated with aging. Curiously, young larvae (<6 hrs old) were less able to develop to mature muscle larvae than older larvae. In contrast to these findings we were unable to find differences in recirculation capacity, or development to mature muscle larvae when comparing larvae of 2 or 24 hours of age. Methodological differences may account for these discrepancies. While the observation that cuticular changes occur during the first 6 hours of independent larval life is interesting and needs to be pursued, there are no indications yet of what purpose or function these changes can have for the larvae. Indeed, it is difficult to relate cuticular molecular variations to larval resistance to host immunity since 50% of larvae disappear from the circulation in a primary infection 2 hours after their production, yet these larvae are the ones that appear most susceptible to immunity; older larvae being conspicuously resistant. Nevertheless, the age-related molecular heterogeneity of the cuticle may well provide important clues to the penetration of muscle and/or subsequent development of the young NBL.

Immunity to NBL exists in vivo and can be extremely effective in preventing NBL from establishing in striated muscle. Immunization is effective with either an intravenous injection of NBL or an oral infection with muscle larvae. In all the above investigations, protection was gauged by muscle larvae burden. This information has set the stage for studies of where and when immunity occurs, particularly in the primary infection, and which mechanisms are effective in vivo. Since NBL begin their systemic migration immediately after their production in the intestine, they are likely to be susceptible to immunity anytime after they are born and any site through which they migrate. This is essentially the whole body. Indeed, our results showed that when we attempted to recover NBL from the peritoneal cavity, portal vein and thoracic duct lymph after challenge infection from previously immunized rats, the time of appearance of these larvae was significantly delayed and the recovery was significantly reduced in all sites examined(76). Obviously, the host immunity not only impeded NBL migration but also effectively destroyed them. Our later study also indicated that this immunity is specific to NBL, irrespective of their age(77). It should be noted that the experiments prior to ours did not exclude the possibility that the protection was also directed against maturing muscle larvae as they developed from the challenge NBL whereas our experiments were specifically designed to study immunity against NBL alone.

Mackenzie et al. (9) studied the kinetics of anti-NBL immunity by examining the capacity of immune serum to mediate cell adherence in vitro. The activity was first found in serum collected 15 days after infection with 4,000 muscle larvae and the highest titer was observed 20 days after infection. Philipp et al.(78)and Jungery and Ogilvie (13), working with rats and mice respectively, found that the antibody-mediated cellular cytotoxic activity in serum was generated 15 days or 25 and 30 days after T. spiralis infection rats or NIH and C₃H mice respectively. Protection was demonstrated in vivo as in Wistar early as 10 days after a single iv injection of NBL in DBA/1 and AKR mice and this immunity was 96 to 98% protective against a NBL challenge(45). Because of the high degree of protection on day 10 it seems likely that NBL induced immunity was function prior to this. More recent data from us show that immunity can be demonstrated in vivo in rats 7 days after the initial infection and gains real strength by Day 10. This suggests that NBL are highly immunogenic and correlates the time of appearance of immunity with the precipitous decline in NBL output seen in vivo (76,77).

Bell et al.(45)provided evidence for nonspecific resistance to NBL in vivo. In these experiments, muscle larvae burdens of various inbred mice were examined after a single iv injection of NBL. A consistent pattern was observed in which C₃H strain mice always harbored the highest and DBA/1 mice the lowest burden of muscle larvae. Since these studies examined a single intravenous encounter with NBL in non immune mice, and the great majority of larvae have left the circulation by 24 hours(58), it is likely that the low muscle larvae burden of DBA/1 mice reflects a nonimmunological resistance to NBL. A comparison of muscle larvae burden with the MHC haplotype showed that k-haplotype mice(C₃H and AKR) had high muscle larvae burdens whereas q-haplotype mice(B10Q and DBA/1) had low burdens. This correlation suggests that components of nonspecific resistance are influenced by the MHC. These results parallel the earlier findings of Wassom et al. (43,44) in MHC association with high (H-2k) and low (H-2q) muscle larvae burdens after a single oral infection with muscle larvae. When muscle larvae burdens after a single injection of NBL were compared with those after a challenge injection with NBL there was a dissociation of "non-specific resistance" and immunity to the challenge. This dissociation of immunity to a challenge infection compared with resistance to a primary infection was interpreted as supporting the concept of a nonspecific resistance that was distinct from acquired immunity.

A considerable body of evidence implicates eosinophils and/or neutrophils in NBL killing. The first suggestion of a functional association between eosinophilia and protection was provided by Grove et al. (112) who showed that anti-eosinophil antisera administered in vivo increased the number of muscle larvae in treated rats but did not affect adult worms. The implied effect on NBL of eosinophils was quickly confirmed in vitro (8,9). Eosinophils appear to require antibody of the IgG class (19) to mediate cellular adherence but at least two processes lead to killing in vitro. The most extensively studied are the products of oxidative metabolism H₂0₂ (peroxide) plus superoxide (0₂^-) and free Cl^- (113,114) which produce up to 100% killing in vitro. The observation that leukocytes from a patient with chronic granulomatous disease (CGD) demonstrated significantly reduced larvicidal activity further suggests that the killing mechanism encompasses an oxidative response by normal leukocytes as granulocytes from CGD patients do not generate hydrogen peroxide (113). In addition, eosinophil major basic protein has also been shown to damage and kill NBL in vitro(115). Cells other than eosinophils also have a role, Mackenzie et al.(114) showed potent killing by neutrophils and Buys et al. (117) found that myeloperoxidase from neutrophils was more effective than eosinophil peroxidase in killing NBL. A variety of cell types can destroy NBL in addition to neutrophils and eosinophils. Mackenzie et al.(102) showed that mast cells, eosinophils, neutrophils and macrophages were all able to adhere to and destroy NBL during in vitro incubation even though significantly different adherence patterns were obtained. Cells do not attach to dead larvae leading to the suggestion that a chemotactic factor may be released from the live parasite which causes cellular adherence(100). Recent evidence shows that intestinal lamina propria cells are enriched with larval killing eosinophils(118), which confirms our finding that NBL are destroyed in significant quantity within the intestinal mucosa.

Evidence for antibody-dependent cell-mediated destruction of NBL is largely based on in vitro studies. In all these experiments it appeared not to matter whether leukocytes, especially eosinophils, were collected from normal hosts (19,72,102,113,116), from animals infected with Schisotosoma mansoni (117), Mesocestoides corti (13,69), or from T. spiralis infected mice(8), rats(120), and from Trichinella infected humans(73,121), as long as immune serum was present in the culture in addition to the cells NBL were killed. The results of Bruschi et al. also demonstrated that the sera factors inhibited neutrophil’s oxidative metabolism, hence suggesting certain cytotoxic mechanisms other than production of superoxide anions may play a role(121).

The role of IgG has been unequivocally, established in a report describing a monoclonal IgG antibody against the 64K antigen of NBL which mediated eosinophil-dependent destruction of this stage in vitro. NBL treated with this monoclonal antibody in vitro and then injected into recipient mice that also received a further passive transfer of the anti-64K mAb for 3 days, showed a 36-51% reduction in muscle larvae burden. Conflicting evidence exists over the role of complement. Kazura and Grove(8)could find no effect but Mackenzie et al.(9)showed an effect of complement and so has a most recent study (62). In vivo studies have been limited, but in addition to the work of Grove et al. (112), described above, Dessein et al.(122) provided evidence in favor of a role for eosinophils and IgE. These authors administered rabbit anti-E chain antibodies to rats to suppress the production of IgE antibody. They then found that after infection with T. spiralis, the rats had reduced mast cell degranulation, decreased eosinophil numbers in the blood and tissues and increased muscle larvae burdens after a complete infection. Administration of IL-3 enhanced the IL-4-dependent IgE response whereas treatment with anti-IL-4 antibody suppressed such response(123,124), leaving no doubt that T cells are involved in the specific immune response. Our own data also have shown that larval migration is blocked irrespective of the site that larvae are introduced into the body. Larvae are promptly killed after intravenous or intraperitoneal injection into rats 15 or 16 days after a primary infection. In addition, if adult worms collected from non-immune rats are transplanted into the duodenum, more than 90% of the NBL produced do not enter draining lymphatics or capillaries (76,125), which is indicative of potent intestinal immunity.

Our electron microscopic studies revealed that in all cases larvae recovered from these local sites are covered with dense clusters of granulocytic cells(125). It appears that immunity, once induced, acts powerfully at any site that larvae are likely to contact in their systemic migration. Apparently the strength of this response is so great that few larvae will escape the confines of the local intestinal environment where they are produced. A most recent study in humans also confirmed this antibody-dependent cell-mediated cytotoxic effect on NBL(121). Obviously, what were discovered in rodents reflected what can take place in humans.

In vivo mechanisms and targets of immunity against NBL are the least understood component of this host parasite interaction. Considering the numerous in vitro descriptions of functional systems it is fair to conclude that NBL may be the targets of a diverse array of host effector processes. Both IgG and IgE antibodies have been implicated directly, while a variety of granulomatous or phagocytic cell types are able to mediate larval killing. Mechanisms of killing currently include oxidative processes and eosinophil major basic protein while a role for complement is also suggested by some studies. When all of these processes have been more precisely measured in vivo it seems likely that the NBL may be the target of more independent immunological and non immunological killing mechanisms than any other stage of the T. spiralis life cycle. Indeed T. spiralis may well be unique in the speed with which each life cycle stage induces immunity and the speed and degree to which the parasite modifies functional antigens during development and differentiation. If these processes are related then it appears that T. spiralis has specialized in a foreshortened life-cycle that remained strongly immunogenic thus outrunning immunity. Immunosuppression, although present(121,126,127), appears to be a secondary and less important process in the overall economy of the infectious event, especially regarding the primary infection.

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