Possible immunological damage to the tegument of Hymenolepis diminuta in mice and rats.

Opaque or darkened areas (DA) of variable size and position occur on Hymenolepis diminuta in mice and rats. In mice DA normally first appear in the neck region of the worm but subsequently they appear elsewhere and increase in number until destrobilation or worm expulsion. The posterior of destrobilated worms is often darkened. In the more immunogenic infections with six cysticercoids there are more DA per worm than in infections with one cysticercoid. DA are areas of the tegument with a homogeneous increase in electron density; abnormal mitochondria; reduced granular endoplasmic reticulum, Golgi complexes and discoidal secretory bodies; and accumulation of lipid droplets. DA disappear from worms maintained for up to 4 h in Hanks' balanced salt solution and can be induced by mechanical damage to the worms. As the numbers of DA increase with the duration and intensity of infection and have similarities with types of cell injury, they are probably sites of worm pathology induced by host immunity.     

Biochemical effects of thiabendazole and cambendazole on Hymenolepis diminuta (Cestoda) in vivo

An investigation of the chemotherapeutic and biochemical effects of two benzimidazole anthelmintics, thiabendazole (TBZ) and cambendazole (CBZ), on Hymenolepis diminuta in experimentally infected rats is reported. Thiabendazole was active against H. diminuta at a relatively high dosage. A single oral dose of TBZ at 250 mg/kg body weight on day 15 of infection eliminated 100% of the tapeworms as determined at necropsy 5 days after treatment. The chemotherapeutic actions of TBZ on H. diminuta were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TBZ 24 hr earlier were significantly smaller and contained much less glycogen (as a percent of the wet weight) than worms from unmedicated controls. Protein concentrations increased in TBZ-treated worms and at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TBZ-treated worms were significantly lower than the corresponding control values. Cambendazole proved to be five times more potent than TBZ against H. diminuta and produced the same basic changes in worm weight and chemical composition within 18 hr of treatment of the host. Administration of a single oral dose of TBZ or CBZ to the host produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm weight and chemical composition. That change, observed in in vitro studies carried out 14 hr after treatment, revealed that tapeworms from drug-treated rats absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability of the worm to accumulate glucose against a concentration difference was significantly depressed.     

Secondary infections of Hymenolepis diminuta in mice: effects of varying worm burdens in primary and secondary infections.

In one (1 c) and six (6 c) cysticercoid primary infections of Hymenolepis diminuta in NIH (inbred) and CFLP (outbred) male mice 6 +/- 1 weeks old greater than 85% of the worms established but were rejected (destrobilated or expelled) subsequently. Rejection occurs more quickly in 6 c infections than in 1 c infections. Considerable worm growth occurs in 1 c and 6 c primary infections but worms from 6 c infections weighed less than worms from 1 c infections on all days studied. Expulsion of H. diminuta does not occur more rapidly in secondary infections than in primary infections; loss of 6 c secondary worms occurs at the same rate as 6 c primary worms but 1 c secondary worms survive longer than 1 c primary worms. Although worms are not lost more quickly in secondary than in primary infections, they are affected at an early age by the immune response which stunts their growth. Increasing the intensity of primary and secondary infections increases the severity of stunting of secondary worms. The results are discussed and it is suggested that immune responses to Hymenolepis spp. in rodents are common but that thresholds of worm numbers exist below which appreciable worm loss does not occur. Stunting due to crowding, which generally is attributed to inter-worm competition, may be in part immunologically mediated. For future immunological studies attempting to induce secondary responses to H. diminuta in mice, worm growth, not survival, is the criterion to evaluate.     

Structure-activity relationships of benzothiazole and benzimidazole anthelmintics: a molecular modeling approach to in vivo drug efficacy

An investigation of the biochemical effects of an anthelmintic, tioxidazole (TIOX, methyl 6-[n-propoxy]benzothiazole-2-carbamate), on Hymenolepis diminuta in experimentally infected rats is reported. The chemotherapeutic actions of TIOX on H. diminuta in vivo were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TIOX 24 hr earlier were significantly smaller and contained much less glycogen (as a percentage of the wet weight) than worms from untreated controls. In TIOX-treated worms, protein concentrations rose at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TIOX-treated worms were considerably lower than the corresponding control-values. Differences in the absolute amounts of glycogen and protein between control and drug-treated worms were even more pronounced. Administration of a subcurative dose of TIOX to the rat produced in H. diminuta another change, the onset of which preceded the gross alterations in worm weight and chemical composition. In vitro studies, carried out 18 hr after treatment, revealed that TIOX-treated worms absorbed and metabolized much smaller quantities of exogenous glucose than did the controls and that the ability of the worm to accumulate glucose against a concentration difference was significantly depressed. A mode of action common to the structurally related benzothiazole and benzimidazole anthelmintics is indicated by the similarity of their biochemical and physiological effects on the tapeworms and their time course of action when administered to rats infected with H. diminuta. Molecular modeling revealed that the benzothiazole and benzimidazole anthelminitics are congruent electronically and structurally. In vivo drug efficacy depends upon the magnitude of the molecular dipole moment and the percentage of polar surface area. Within the benzimidazole series, structural and electronic congruence is found between the 2-thiazolyl and 2-methyl carbamate groups, suggesting that these groups behave similarly in transport to, and binding at, the active site. Finally, anthelmintics that have the 5' substituents twisted out-of-plane were more active than those anthelminitics with 5' substituents in-plane. All of these factors implicate a highly polar, L-shaped cleft to which the anthelmintics bind at the active site.     

Trichinella spiralis: mediation of the intestinal component of protective immunity in the rat by multiple, phase-specific, antiparasitic responses.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.     

Immunity to adult cestodes: basic knowledge and vaccination problems. A review.

Immunity in mammals to intestinal cestodes has been reviewed using the normal final host infected with the tapeworms Hymenolepis diminuta in rats and H. microstoma and H. nana in mice as a model. Primary infections up to a certain level continue to live as long the host, while most worms in infections with larger doses are destrobilated and expelled. It has been argued that concomitant immunity against a superimposed infection exists in rats and mice infected with H. diminuta and H. microstoma, respectively, and suggested that it also takes place in humans infected with Taenia spp. Immunity to secondary infections after expulsion of a primary infection occurs, but immunological memory is rather short-lived, although depression of worm growth occurs for at least two third of the rat's life. Serum antibodies have been shown to produce a direct precipitate on the surface of cestodes in vitro, but a direct effect of antibodies in vivo or the relationship with e.g. host effector cells, like mast cells and eosinophils, is unknown. It has been shown that peritoneal exudate cells from rats are able to kill H. diminuta in vitro. Very little is known about the mechanisms of tapeworms to counteract host immunological responses, but the tegumental glycoconjugates and discoidal secretory bodies are possible candidates. Passive transfer of immunity by mesenteric lymph node cells has only been successful using cells from H. nana egg-infected mice and has shown that only short-lived proliferating cells are responsible for transferring immunity. Vaccination procedures and problems are discussed with special reference to E. granulosus in dogs.     

Study of the effects of insulin on the migration of Hymenolepis diminuta in rats

The effects of insulin on worm (Hymenolepis diminuta) migration was studied. Insulin injection (20 U/kg, s.c.) significantly increased gastric acid output but did not affect the serotonin content of blood, intestinal lumen or worms. The drug produced, dose-dependently, posteriad migration of the worms in rats without pylorus-ligation but ligation of the pylorus prevented this migration. It is concluded that the hypersecretion of gastric acid induced by insulin is responsible for the posteriad migration of H. diminuta in rats.     

Hymenolepis diminuta: lack of sulphate activation and sulphotransferase activity

There is no evidence that Hymenolepis diminuta can carry out sulphoconjugation reactions. Neither whole worms nor worm extracts were able to sulphate 4-methylumbelliferone. No sulphotransferase activity could be demonstrated in H. diminuta using a variety of substrates, nor was H. diminuta capable of synthesising the sulphate donor 3'-phosphoadenosine-5'-phosphosulphate from ATP and inorganic sulphate. Possible alternative sources of active sulphate in this parasite are discussed.     

Inhibition of growth of a tapeworm Hymenolepis diminuta in its normal host (rat).

The biomass of 8-day-old worms of Hymenolepis diminuta in secondary infections, administered to rats 3-10 days after chemotherapeutically expelling a primary infection, was 70-90% less, and the worms were more posteriorly distributed, than in naive controls. The strong depressive effect on growth waned rapidly over 2-5 weeks, but even in rats not challenged until 17 months later, worm growth was weakly depressed by 30%. The extent to which growth was depressed in a secondary infection was independent of the number of worms in the challenge but increased with number of worms in the immunizing infection up to four to eight worms. Further increase up to 64 worms had little effect. This suggests, as it is known that the biomass of worms in a rat reaches a maximum with infections of between five and 10 worms, that the change in the intestine is proportional to biomass, not number, of worms. It is argued that partially suppressed immuno-inflammatory changes in the intestine, which will affect secondary worms so strongly, will also have depressed growth and fecundity effects on the primary worms, that a dynamic equilibrium is reached between the strength of the intestinal response and the biomass of the tapeworm, and that it is reaching this equilibrium, not a 'crowding effect', which limits H. diminuta to a level compatible with the survival of the rat.     

Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains.

Five-week-old DA male rats infected with 10 Hymenolepis diminuta cysticercoids showed significant mastocytosis 6 weeks post-infection and low persistence of worms. In F344/N rats, however, no mastocytosis and no worm loss occurred during a 6 week infection. Mucosal mast cells appear to be associated with the expulsion of H. diminuta from DA rats.     

Scanning electron microscope studies of the mucosa of rats infected with Hymenolepis diminuta (Cestoda)

The intestinal morphology of rats given one, 10 or 100 cysticercoids of hymenolepis diminuta was examined by scanning electron microscopy. The presence of this tapeworm causes extensive villous atrophy and fusion. The most extreme changes in mucosal architecture were observed adjacent to the mature proglottides of the worm and in these areas the villi were reduced either to flattened plate-like structures or to low irregularly shaped undulations. The presence of one large H. diminuta resulted in more severe pathological damage than caused by several smaller worms. Colonization of the upper region of the ileum by long filamentous bacteria was also observed in rats infected with H. diminuta.     

Effect of host feeding and available glucose on glycogen synthase and phosphorylase activities in Hymenolepis diminuta and Vampirolepis microstoma

The influences of host feeding and the availability of glucose in vitro on the activities of glycogen synthase and glycogen phosphorylase in Hymenolepis diminuta and in Vampirolepis microstoma were studied. The worms were recovered from hosts that had been fed ad libitum, starved for 24 hr, or starved 24 hr and then refed for 1 hr immediately prior to worm recovery. The ratios of active to inactive glycogen synthase and phosphorylase were correlated with the host feeding regimen prior to recovery. Glycogen synthase in H. diminuta was predominately in the inactive D form in worms from both fed and fasted hosts. One hour after refeeding, up to 80% of the synthase was in the active I form. Phosphorylase in H. diminuta was predominantly in the active a form in worms from fed and fasted hosts, but activity of this enzyme was suppressed in worms from refed hosts. When H. diminuta from fasted hosts was incubated in a balanced salt solution containing 40 mM glucose, glycogen synthase I increased, and phosphorylase a decreased. Glycogen synthase in V. microstoma was predominantly in the inactive D form in worms from both the fed and fasted hosts, but the proportion in the active I form increased to over half the total synthase by 1 hr of host refeeding. The proportion of glycogen phosphorylase a was high in worms from fed hosts and decreased, but not dramatically, in worms from fasted hosts. The results suggested that the worms had access to another source of glucose, probably from the host bile, and we measured a low but significant concentration of carbohydrate in the gall bladder bile of mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro tapeworm extract-induced proliferative responses of gut-associated lymphoid cells from Hymenolepis diminuta infected mice

The development of lymphoid cells reactive to tapeworm-associated antigens during the course of Hymenolepis diminuta rejection from mice was studied using an in vitro tapeworm extract (TWE)-induced cell proliferation culture system. Mice infected with three cysticercoids on day 0 developed three adult worms by day 7 but worms were rejected by day 21 post-infection. Concomitant with worm rejection was the development of TWE-sensitized lymphoid cells which responded by proliferation when stimulated in vitro with TWE. Sensitized cells were detected in gut-associated mesenteric lymph nodes but were not detected in spleen, axillary lymph nodes, or Peyer's patches of infected mice, or in lymphoid organs of non-infected mice. These studies suggest that rejection of H. diminuta from mice is associated with the activities of gut-associated, tapeworm antigen-sensitized immune cells localized in the mesenteric lymph nodes.     

Some influences of population density on Hymenolepis diminuta in rats.

When measured 56 days postinfection the length, wet weight and dry weight of Hymenolepis diminuta were all found to decrease with increasing number of cysticercoids given up to 20. The mean position of the worms in 10, 12 and 20 worm infections is significantly posterior to that of 1, 2 and 5 worm infections and the worms are attached over a wider area of the intestine. Egg production by the worms was followed up to day 56 postinfection; the number of eggs produced per worm and even per rat decreased with increasing population density. Thus the best way to get most eggs and to maintain the parasite in the laboratory is to have rats infected with only one tapeworm. Rats given 1-20 cysticercoids showed a mean recovery of 100-65%, while rats given 40-200 cysticercoids showed a mean recovery ranging from 13 to 2%. In addition to 'normal' worms, defined as worms greater than 10 mm, small, most probably destrobilated, worms were found. In the 50 and 100 cysticercoid infections, worm recoveries were, respectively, 8% 'normal', 16% small, and 2% 'normal', 5% small. From the significantly lower recovery from heavy infections it is concluded that a deleterious factor is operating during the 8 weeks after the infection.     

Glycogen synthase in the rat tapeworm, Hymenolepis diminuta--I. Enzyme activity during development and with crowding.

1. Activity of glycogen synthase (E.C. 2.4.1.11) in Hymenolepis diminuta (Cestoda: Cyclophyllidea) was investigated as a function of development and with crowding. 2. Synthase activity was low in the anterior and posterior ends of the worms and highest in the pregravid proglottids in the mid-portion of the strobila. 3. The enzyme activity increased during development of the cestode at least up to 15 days postinfection, but the increase in activity apparently was not due to conversion of the inactive to the active form. 4. Mature oncospheres also contained glycogen synthase, but the activity was lower than in strobilar tissues. 5. Synthase I activities and the proportion of total activity in the I form were generally higher in worms from high density (100 worm) infections than in those from low density (10 worm) infections.     

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

The occurrence and distribution of 5-hydroxytryptamine in Hymenolepis diminuta and H. nana

The presence of 5-HT in Hymenolepis diminuta and Hymenolepis nana was detected by 2 biochemical methods and as yellow fluorescence in a histochemical method. In H. diminuta, 5-HT was found in a concentration of about 1.2 micron/g; this amount did not vary significantly in worms aged 6 to 18 days or more or in various regions of the worm. In H. nana, 5-HT was found in a concentration of about 1.8 micron/g. It was histochemically localized in H. diminuta and H. nana in a pattern similar to that of acetylcholinesterase previously described in these 2 cestodes, and it may be the opposing neuro-transmitter to acetylcholine. The lack of 5-HT in the vestigial rostellum of H. diminuta may be correlated with loss of function of this organ.     

In vivo effects of putative crowding factors on development of Hymenolepis diminuta.

During in vitro incubation, Hymenolepsis diminuta secretes substances into the medium that inhibit DNA synthesis in the germinative region of freshly isolated, uncrowded worms. Of the many substances that are released by H. diminuta into the medium, earlier studies indicate that only succinate, acetate, glucosaminic acid, and cGMP are responsible for the inhibition. In the present report, effects of these putative crowding factors on worm development in vivo were examined. At 7 days postinfection the proximal end of the host's intestine was catheterized and perfused with test solution. The test solution contained 28 nM cGMP, 250 microM glucosaminic acid, 120 mM succinate, and 40 mM acetate. The solution was perfused by a peristaltic pump at a rate of 50 ml/day. At 2 wk postinfection, worms were recovered for subsequent analysis. Worms developing in the presence of crowding factors were 53% less in wet weight than control worms. Carbohydrate concentrations in worms from experimental groups were not different from those in control groups; therefore, the inhibition in growth was probably not due to carbohydrate deprivation. Worms from experimental groups had fewer immature, mature, and gravid proglottids than did worms from control groups. The results are consistent with the hypothesis that the tested substances, which inhibit DNA synthesis in H. diminuta in vitro, are a part of the cause of the crowding effect in vivo.     

Biochemical effects of fenbensazole on Hymenolepis diminuta in vivo

An investigation of the chemotherapeutic and biochemical effects of a new broad-spectrum anthelmintic, fenbendazole, (FBZ, methyl 5-[phenylthio]-benzimadazole-2-carbamate), on Hymenolepis diminuta in experimentally infected rats is reported. FBZ proved to be highly active against H. diminuta; a single oral dose of 12.5, 25 and 50 mg/kg body wt. on day 15 of infection eliminated 77, 100 and 88% of the tapeworms respectively as determined at necropsy 24 h after treatment. The chemotherapeutic actions of FBZ on H. diminuta in vivo were accompanied by marked changes in worm wt. and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of FBZ 18 h earlier were significantly smaller and contained much less glycogen (as a percent of the fresh wt.) than worms from unmedicated controls. Protein concentrations rose in FBZ-treated worms, but more slowly than the rate of decline in glycogen concentration. Glycogen/protein ratios in FBZ-treated worms were considerably lower than the corresponding control values. Differences in the absolute amounts of glycogen and protein between control and drug-treated worms were even more profound. Administration of a single oral dose of FBZ (14 mg/kg) to the rat produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm wt. and chemical composition. In vitro studies, carried out 16 h after treatment, revealed that FBZ-treated worms absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability to the worm to accumulate glucose against a concentration difference was significantly depressed.     

The fecundity of Schistosoma mansoni in chronic nonhuman primate infections and after transplantation into naive hosts

Adult Schistosoma mansoni worms were transplanted from 8 nonhuman primates with chronic infections into 8 naive recipients, in an effort to test the hypothesis that worm fecundity reduction in chronic infections is the result of host immunity or some other host effect. Techniques for perfusing living donors without the added use of anti-schistosomal drugs and for reducing the likelihood of post-operative bacterial endotoxemia and septic shock are described. Fecundity values in terms of eggs per day per female worm were obtained for the worms in their original and in their new hosts and compared. In 3 experiments, perfusions were incomplete and the donors were saved, enabling direct comparisons of fecundity to be made in subpopulations of worms in both their original and new hosts, after equal life spans. In only 1 of the 8 transplantations was there a clear increase in fecundity after surgical introduction into a naive host. Therefore, these experiments fail to support the hypothesis that reduced fecundity of S. mansoni worms in permissive nonhuman primate hosts is a reversible result of host immunity or some other host-derived factor. Despite this negation, further evidence for reduced worm fecundity in older infections was obtained. In the absence of in vivo evidence for immune-mediated antifecundity, worm senescence is the most likely explanation for this finding, with irreversible immune damage to the worms being a less attractive alternative hypothesis.