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.     

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)     

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.     

Glycogen synthase in the rat tapeworm, Hymenolepis diminuta--II. Control of enzyme activity by glucose and glycogen.

1. The proportion of activity in the physiologically active I form of glycogen synthase in Hymenolepis diminuta (Cestoda) decreased in the worm when the rat host was fasted and was greatly increased in the cestode 1 hr after a 24 hr fasted rat was refed. 2. The increase in glycogen synthase I activity was due to glucose present in the host gut after feeding, not to other physiological changes in the rat intestine due to meal consumption. 3. Incubation of intact H. diminuta in vitro with glucose also resulted in the conversion of glycogen synthase D to I. 4. Glucose does not appear to affect the glycogen synthase complex directly, because neither the total synthase converted to I nor the rate of conversion was affected by glucose in a partially purified homogenate. 5. High concentrations of glycogen inhibited the synthase D to I conversion and high mol. wt glycogen was a more effective inhibitor than low mol. wt glycogen.     

Experimental and theoretical studies of albendazole, oxibendazole, and tioxidazole

An investigation of the therapeutic effectiveness of albendazole (ABZ, methyl 5-[propylthio]benzimidazole-2-carbamate), oxibendazole (OBZ, methyl 5-[n-propoxy]benzimidazole-2-carbamate), and tioxidazole (TIOX, methyl 6-[n-propoxy]benzothiazole-2-carbamate) against Hymenolepis diminuta in experimentally infected rats is reported. All of the anthelmintics tested were effective therapeutically as a single oral dose against adult tapeworms, however, at different dose levels. The rank order of in vivo anthelmintic potency was ABZ greater than OBZ greater than TIOX. Molecular modeling revealed that drug efficacy depends on the orientation of the propyl group at position 5 on the heterocyclic ring system and on the magnitude of the molecular dipole moment.     

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.     

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.     

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.     

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.     

Developmental physiology of cestodes: cyclic nucleotides and the identity of putative crowding factors in Hymenolepis diminuta

Worm-conditioned saline (WCS) was prepared by incubating Hymenolepis diminuta from crowded infections for 12 hr in a balanced salt solution. The effect of the WCS on the incorporation of [3H] thymidine into DNA in the anterior regions of fresh H. diminuta was compared to effects produced by the cyclic nucleotides in the WCS. Cyclic AMP and cGMP were found in the WCS, and cGMP but not cAMP (at the concentration in WCS) caused some inhibition of DNA synthesis. For further study of the effects of cyclic nucleotides, worms were incubated with theophylline, caffeine, 3-isobutyl-1-methyl xanthine, 2-deoxy cGMP, and L-ascorbic acid, all of which produced some inhibition of [3H] thymidine incorporation. Treatment of WCS with 3',5' cyclic nucleotide phosphodiesterase abolished part of its inhibitory activity, i.e., that part presumed to be due to cGMP. When worms were incubated in the presence of succinate, acetate, D-glucosaminic acid, and cGMP simultaneously and in the concentrations each was found in the WCS, DNA synthesis was inhibited to a degree equal to that found in the WCS. Thus these substances apparently represent the putative crowding factors in the WCS. WCS prepared with worms from different population densities contained the same levels of cAMP but varied in content of cGMP, which decreased as the worm density increased. WCS prepared with patent worms contained high levels of cAMP, but the same amounts of cGMP as WCS prepared with 10-day-old worms. At least some inhibitors of cyclic nucleotide phosphodiesterase inhibited the secretion of cGMP by the worms. Levels of cGMP in the host intestine varied with the presence or absence of worms, number of worms, and area of the intestine.     

Hymenolepis diminuta: immunogenicity of the strobilate worm

Mice were immunized against challenge with Hymenolepis diminuta by feeding cysticercoids or by surgically implanting into the duodenum strobilate worms of different ages. Young worms stimulate stronger immunity than older ones, although the latter presents the host with a greater amount of strobilar tissue per unit time. An increase in the number of immunizing worms is associated with an increase in the level of protection. It is concluded that the development of functional immunity against H. diminuta in mice has both quantitative and qualitative antigenic requirements; it is influenced by worm age and is independent of worm mass.     

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.     

Differential establishment and survival of Hymenolepis diminuta in syngeneic and outbred rat strains.

Experimental Hymenolepis diminuta infection was carried out in inbred strains of rats (F344/N, JAR-2, LOU/M, TM, DA and DA-bg/bg) and outbred Wistar rats. All strains became infected with this cestode, but clear strain-dependent variation in the susceptibility to H. diminuta infection was observed. Marked differences in worm persistence and worm weight were found at 6 weeks post-infection in TM and DA rats. These strains would be useful to clarify the interactions between H. diminuta and its rat host.     

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.     

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.     

Studies on the reproductive system of Hymenolepis diminuta using autoradiography and transplantation.

Adult Hymenolepis diminuta exposed in vitro for 3 hr to 3H-thymidine showed incorporation of the isotope on autoradiograms over nuclei of actively dividing cells in the testes, ovary, vitellaria, and developing embryos in the eggs. Timing studies utilizing labeled worms transplanted to uninfected hosts showed that it took 18 hr for spermatogonia to develop to primary spermatocytes, 24 hr to secondary spermatocytes, 36 hr to spermatids, and 48 hr to sperm bundles. Self-insemination was confirmed in single worm transplants of 3 days by the presence of labeled sperm in the seminal receptacles. In multiple worm transplants labeled worms inseminated themselves in each case and cross-inseminated with 92% of the unlabeled worms present.     

Hymenolepis diminuta: effects of amoscanate on energy metabolism and ultrastructure

Amoscanate possesses chemotherapeutic activity against schistosomes, and in higher doses against many other helminths including filariids and Hymenolepis diminuta. The primary mode of action of this compound is unknown. Effects of the drug on the carbohydrate metabolism as well as on the tegumental and nephridial epithelia of H. diminuta were examined. At various time intervals after administration of the drug to rats infected with H. diminuta, the parasites were recovered and incubated in glucose-salts medium for 90 min. Chemotherapy resulted in decreases in succinate, lactate, and acetate recoveries, while ATP levels dropped. In addition, glycogen levels were depressed in drug-treated worms which were homogenized immediately upon isolation. Glycogen synthase I activity was inhibited 16-61% in cestodes obtained from Amoscanate-treated animals and homogenized immediately, but returned to normal levels after incubation for 90 min in glucose-salts medium prior to homogenization and assay. Phosphorylase a activity was found to be 25-30% higher in preparations of worms from drug-treated rats, which correlates with the rapid depletion of glycogen in parasites exposed to the drug. However, in contrast with glycogen synthase activity, the elevation of phosphorylase a activity in H. diminuta exposed to the drug was not readily reversible. Attempts to demonstrate activity of the drug in vitro by incubating intact cestodes directly with Amoscanate were unsuccessful. Thin sections of parasites obtained from Amoscanate-treated rats and examined by transmission electron microscopy revealed surface alterations of the tegument and nephridial canals. Alterations included bleb formation and erosion of microtriches from the tegument, as well as disappearance of microvilli from nephridial canals. However, these effects became manifest only after 4 or more hr exposure of the rat to the drug. Biochemical effects, on the other hand, were significant after 3 hr exposure.     

Developmental physiology of cestodes. XIV. Roughage and carbohydrate content of host diet for optimal growth and development of Hymenolepis diminuta.

Diets of rats infected with Hymenolepis diminuta (CESTODA: Cyclophyllidea) were altered with respect to carbohydrate content and to roughage, and the effects on worm growth and development were studied. Compared to worms from rats fed a 56% glucose diet, those on a 56% starch diet were heavier at 10 and 15 days and had more immature proglottids at 5 days, mature prglottids at 10 days, and mature and gravid proglottids at 15 days postinfection. In addition, worms from rats fed the starch diet contained a higher carbohydrate concentration and a lower lipid concentration from those fed the glucose diet. Worms from rats fed diets with combinations of carbohydrates such as 51% starch-5% sucrose and 51% starch-5% lactose were not different from those fed the 56% starch diet. If rats were fed a pellet diet (Purina Laboratory Chow), the worms grew substantially larger than those from rats fed the 56% starch or combination diets. The differences could be overcome if a 6% roughage component were included in the 56% starch diet. Therefore, the starch-roughage diet here presented is recommended as the optimal defined diet for studies of the development of H. diminuta in the definitive host.     

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.     

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.