The experimental infection of pigs with different numbers of Taenia solium eggs: immune response and efficiency of establishment

Three of 4 pigs inoculated with 10 eggs of Taenia solium became infected. In those pigs infected with larger numbers of eggs, all became infected. Specific antibodies against the metacestodes were found in serum at day 30 postinoculation (PI) in animals that received 1,000 or more eggs and at day 60 in those that received 10 or 100 eggs. The concentration and diversity of antibodies increased up to the day of death in pigs that received 10,000 or 100,000 eggs. All pigs infected with 1,000 or more eggs developed antibodies, but only 40% and 75% of pigs that received 10 and 100 eggs, respectively, developed antibodies. Metacestodes were found in the muscles of 23 of the 27 infected animals. In 35.7% of the pigs that received 1,000 or more eggs, metacestodes were also found in the brain. Most of the metacestodes found in pigs infected with 10 or 100 eggs were caseous, whereas in pigs infected with 1,000 or more eggs the majority of metacestodes were vesicular. This study shows that the severity of T. solium infection and the possible regulation of the immune system-evasion mechanisms depend on the number of metacestodes that succeed in establishing themselves and remain vesicular.     

Experimental infection of pigs and cattle with eggs of Asian Taenia saginata with special reference to its extrahepatic viscerotropism.

Asian Taenia saginata, tentatively called Taenia saginata taiwanensis, has been described to be infected in its metacestode stage only in the liver of intermediate host animals. Experimentally, however, we found that the metacestodes of the Asian Taenia saginata are also infected in other viscera than the liver of pigs (Landrace-Duroc-Hampshire) 4 days to 4 months postinoculation (PI). Viscerotropism of cysticercosis was apparent because a majority (70.7%) of the non-calcified cysticerci were found in the livers while a minority were found in extrahepatic organs such as the omentum (19.2%), lungs (8.1%) and serosa of colon (2.0%). When experimentally infected to cattle, Asian T. saginata cysticerci were also observed calcified in the livers. On the other hand, classical Taenia saginata metacestodes infected the muscles and viscera of the Holstein-Friesian cattle whereas no infection was observed in experimental pigs. Extrahepatic metacestodes of Asian T. saginata, which were obtained from an experimental pig were confirmed to be infective to a male volunteer. This extrahepatic viscerotropism of Asian T. saginata metacestodes in experimental pigs explains well the transmission modes of Asian T. saginata among people considering the eating habits.     

Common host antigens in laboratory rats infected with the metacestodes of Taenia crassiceps.

Laboratory rats were infected by intra-peritoneal injection with the metacestodes of Taenia crassiceps. A soluble extract was prepared from the metacestodes removed from the peritoneal cavity of the rats. Double-diffusion, immuno-electrophoresis and fluorescent labelled antibody staining techniques were used. The extract tested against rabbit anti-normal rat serum was found to contain an antigen common to both the host and the parasite.     

Experimental human infection with Asian Taenia saginata metacestodes obtained from naturally infected Korean domestic pigs.

The infectivity of metacestodes of Asian Taenia saginata, now tentatively called Taenia saginata taiwanensis, in human host was confirmed. The metacestodes used in experimental infection were collected from the livers of naturally infected domestic pigs at an abattoir in Cheongju City, Korea. The first gravid proglottid was spontaneously discharged 76 days after infection. Two worms were recovered two years later by chemotherapy. The scolex was unarmed. The number of main uterine branches, varying from 16 to 21, was similar to that of classical Taenia saginata. The liver of pigs was confirmed to be an infection source of Asian T. saginata in Korea.     

Life cycle and postembryonic development of Oochoristica anolis (Cyclophyllidea: Linstowiidae)

Gravid proglottids of Oochoristica anolis from naturally infected anole lizards, Anolis carolinensis, were placed in covered Petri dishes with laboratory-reared beetles, Tribolium confusum and Tenebrio molitor. After maintenance at 25 C, metacestodes developed in 29 of 61 T. confusum (48%), but in none of 5 T. molitor. Beetles contained from 1 to 22 metacestodes (means = 3.3), which were fully developed by day 40 postexposure. A primary lacuna was never observed, but the possibility of its presence could not be ruled out without histological study. No cercomer was formed and metacestodes retained larval hooks throughout development. Scolices were invaginated at removal from the hemocoel, but usually evaginated quickly in Ringer's. On day 60 postexposure, metacestodes were fed by stomach tube to 5 anoles, 2 lacertid lizards (Podarcis muralis) and 2 mice. Worms developed only in anoles, 3 of which were infected upon examination. Oncospheral hooks were present in worms after 7 days development in the lizard; a median excretory pore was present at the posterior tip of all stages examined, including the terminal mature proglottid of a worm after 105 days in a lizard. Scolex growth rate was linear throughout metacestode and adult development, but growth rate in body length was diphasic, punctuated by change of hosts, associated with strobilization. Attempts to establish parenteral infections in anoles were unsuccessful. Present data constitute the most complete life history study thus far for any species of Oochoristica.     

New distribution records of Echinococcus multilocularis in the brown lemming from Barrow, Alaska, USA

We identified Echinococcus multilocularis for the first time in brown lemmings (Lemmus trimucronatus) from Barrow, Alaska, USA. Of 467 brown lemmings trapped between 1995 and 2000, two males and two females (0.9%; 95% confidence interval=0.9+/-0.9%) were found to be infected with metacestodes of E. multilocularis. No metacestodes were found in 17 collared lemmings (Dicrostonyx rubricatus) also trapped at Barrow. In humans, E. multilocularis causes alveolar echinococcosis, which is potentially fatal. Knowledge of the distribution of this parasite is important to protect the public health.     

Asynchronous capsule formation in the gastrointestinal tract of the prairie rattlesnake (Crotalus viridis viridis) induced by Mesocestoides sp. tetrathyridia

The prairie rattlesnake (Crotalus viridis viridis) was experimentally infected with tetrathyridia of Mesocestoides sp. Individual snakes were killed at 4 wk increments, and sections of the stomach, small intestine, large intestine and attached mesenteries were examined for nonencapsulated and encapsulated tetrathyridia. Capsule formation was asynchronous with 9 to 80% encapsulated metacestodes. The distribution of tetrathyridia in the wall of all segments of the gastrointestinal tract is presented as evidence that this metacestode is principally a tissue dwelling parasite.     

Profound Activity of the Anti-cancer Drug Bortezomib against Echinococcus multilocularis Metacestodes Identifies the Proteasome as a Novel Drug Target for Cestodes

A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC₅₀ of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.     

Characterization of the laminated layer of in vitro cultivated Echinococcus vogeli metacestodes

The metacestode (larval) stages of the cestode parasites Echinococcus vogeli and E. multilocularis were isolated from the peritoneal cavity of experimentally infected C57BL/6 mice and were cultured in vitro for a period of up to 4 mo under conditions normally applied for the in vitro cultivation of E. multilocularis metacestodes. In contrast to E. multilocularis, E. vogeli did not exhibit extensive exogenous budding and proliferation but increased in size with a final diameter of up to 10 mm. Most metacestodes contained protoscoleces, singly or in groups, either associated with brood capsules or growing directly out of the germinal layer. Each individual metacestode was covered by an acellular translucent laminated layer that was considerably thicker than the laminated layer of E. multilocularis metacestodes. The ultrastructural characteristics, protein content, and carbohydrate composition of the laminated layer of in vitro cultivated E. vogeli and E. multilocularis were assessed using transmission electron microscopy, lectin fluorescence labeling, and lectin blotting assays. The laminated layer of E. vogeli is, as previously described for E. multilocularis metacestodes, largely composed of N-acetyl-beta-D-galactosaminyl residues and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1,3-galactose. However, in contrast to E. multilocularis, N-linked glycopeptides and alpha-D-mannosyl and/or glucosyl residues were also associated with the laminated layer of E. vogeli. The laminated layer from both species was isolated from in vitro cultivated metacestodes, and the purified fractions were comparatively analyzed. The protein:carbohydrate ratio (1:1) was similar in both parasites; however, the protein banding pattern obtained by silver staining following sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested intrinsic differences in protein composition. A polyclonal antiserum raised against the E. multilocularis laminated layer and a monoclonal antibody, G11, directed against the major E. multilocularis laminated layer antigen Em2 did not cross-react with E. vogeli, indicating distinct compositional and antigenic differences between these 2 parasites.     

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC₅₀ value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.     

Effect of parenterally injected benzimidazole compounds on Echinococcus multilocularis and Taenia crassiceps metacestodes in laboratory animals.

In mice infected with metacestodes of Taenia crassiceps, the following compounds were at least partially effective when injected intraperitoneally at the dosage indicated: cambendazole (500 mg/kg), mebendazole (6.25 mg/kg), oxibendazole (500 mg/kg), 5-benzamido-2(4-thiazolyl)benzimidazole (500 mg/kg), 2-carboethoxyamino benzimidazole (125 mg/kg), and 2-carbomethoxyamino benzimidazole (500 mg/kg). The following were inactive at the dosage indicated: parbendazole (500 mg/kg), thiabendazole (1,000 mg/kg), and fenbendazole (1,000 mg/kg). Mebendazole, which showed some activity at 6.25 mg/kg, was highly active as a single intraperitoneal dose at 25 mg/kg. When injected subcutaneously, mebendazole was much less active than when given intraperitoneally. In mice infected with metacestodes of Echinococcus multilocularis, intraperitoneal injection of mebendazole at 75 to 150 mg/kg, daily for 3 days, was highly effective (95 to 100% reduction in cyst mass). In contrast, oral administration at 1,000 mg/kg, daily for 3 days, was only partially effective. The drug was also effective when given intraperitoneally to infected cotton rats. A water-soluble benzimidazole, carboxymethyleneamino cambendazole, was approximately 50% effective in mice when injected daily for 3 days at a dosage of 75 or 150 mg/kg. The results suggest that, in metacestode infections of medical importance, it may be possible to kill the parasite by delivering a drug to its immediate vicinity, and so to reduce the required dosage with respect to the host.     

Taenia taeniaeformis: inhibition of rat testosterone production by excretory-secretory product of the cultured metacestode

In 3- to 5-month-old male Sprague-Dawley rats infected with the hepatic metacestode, Taenia taeniaeformis, the serum testosterone level was significantly lower than in comparable uninfected controls. By transmission electron microscopy, testicular Leydig cells of infected rats had less smooth endoplasmic reticulum than control Leydig cells. Cultured metacestodes isolated from the hepatic cysts secreted or excreted substances into the incubation medium. The effect of the excretory-secretory product on testosterone concentration in the sera and testes of 15-day-old rats was examined. Subcutaneous injection of 50-200 micrograms of excretory-secretory product/0.1 ml saline/rat for 2 days significantly reduced human chorionic gonadotropin-stimulated serum and testicular testosterone concentrations. Furthermore, the effect of the excretory-secretory product on isolated rat Leydig cell testosterone production was examined. Rat Leydig cells produced testosterone in vitro and, in the presence of 50 IU human chorionic gonadotropin/ml incubation medium, they responded with approximately 100% increase in testosterone production. Addition of 2-10 micrograms excretory-secretory product protein/ml of culture medium significantly reduced the testosterone production by rat Leydig cells in vitro. These results indicate that excretory-secretory product of cultured T. taeniaeformis metacestodes has a direct inhibitory effect on Leydig cell testosterone production under stimulation with human chorionic gonadotropin.     

The nature of antigens common to both the metacestodes of Taenia crassiceps and its laboratory host

Metacestodes of Taenia crassiceps were cultured in laboratory rats and mice, washed in glycine/hydrochloric acid (Gly/Hcl) buffer pH 2.4 and phosphate buffered saline (PBS) pH 7.2 and a soluble extract of the metacestodes (SEM) was prepared from larvae washed in both buffers. The SEM and the original washings were examined for host IgG and a common host antigen (CHA). CHA was present in both the SEM and washings but IgG only in the washings and, in metacestodes from mice only in the Gly/HCl washings). Metacestodes washed in Gly/HCl were not viable. Metacestodes were also maintained in vitro in a peptide nutrient agar and 0.9% saline at 37 degrees C and a SEM was prepared from these; metacestodes from mice had neither host IgG nor CHA but those from rats had CHA but not IgG. SEM prepared from 'normal' metacestodes showed that the CHA consisted of at least two entities, only one having a common identity with the IgG. These results suggest that molecules of both host IgG and a common host antigen are present on the tegument of these parasites.     

Metacestode-induced depression of the production of, and response to, sex pheromone in the intermediate host Tenebrio molitor

Hymenolepis diminuta infection of Tenebrio molitor is associated with an impairment of vitellogenesis and a reduction in host fecundity. In this communication the effect of infection upon an additional aspect of host reproduction, the initiation of mating behavior, has been examined. Copulatory release pheromone, extracted from control virgin females 6-7 days old, was shown to stimulate a positive mating response in 88% of 5- to 6-day-old control males; however, only a 56% response was elicited by pheromone from infected females. In addition, parasitization adversely effected male response to pheromone from control females. A significant (P less than 0.001) depression of copulatory response occurred in infected 6- to 7-day-old males (age of peak response) although this effect was not sustained in older beetles. The possibility that an endocrine interaction between metacestodes and host may mediate these effects is discussed in the light of our knowledge of the role of host juvenile hormone in controlling both pheromone production and vitellogenesis in T. molitor.     

Human Neurocysticercosis Case and an Endemic Focus of Taenia solium in Lao PDR

A male patient with neurocysticercosis was identified in Montai Village, Xay District, Oudomxay Province, Lao PDR in February 2004. He had a history of diagnosis for neurocysticercosis by a CT scan in Thailand after an onset of epileptic seizure in 1993. A pig in the same district was found to contain Taenia solium metacestodes (=cysticerci); the slaughtered pig body contained more than 2,000 cysticerci. In addition to morphological identification, molecular identification was also performed on the cysticerci by DNA sequencing analysis of the mitochondrial cox1 gene; they were confirmed as T. solium metacestodes. The patient is regarded as an indigenous case of neurocysticercosis infected in an endemic focus of T. solium taeniasis/cysticercosis in Oudomxay Province, Lao PDR.     

Ants as first intermediate hosts of Mesocestoides on San Miguel Island, USA

This study tested the hypotheses that ants (Formicidae) function as a first intermediate host of Mesocestoides (Cestoda: Mesocestoididae) and that deer mice (Peromyscus maniculatus) develop metacestode infections after ingesting cysticercoid or procercoid-infected ants. Field studies were conducted at an island fox (Urocyon littoralis littoralis) breeding facility located on San Miguel Island, California Channel Islands National Park, USA, where > 40% of captive foxes were infected with adult Mesocestoides. Eight percent (8%) of deer mice at the fox pen site were infected with Mesocestoides metacestodes while none were infected at a distant site where foxes were absent (campground), thereby indicating the potential localized presence of a first intermediate host. To test whether ants from San Miguel Island contained Mesocestoides DNA, a polymerase chain reaction (PCR)-based diagnostic assay was developed using nested primers that could detect a single hexacanth larva within pooled samples of ten ants. Ants (Lasius niger and Tapinoma sessile) collected near the fox breeding facility were tested using the nested-PCR assay. Seven of 223 pooled samples of L. niger (3.1%) and 2 of 84 pooled samples of T. sessile (2.4%) tested positive for Mesocestoides DNA, while none of the ants were positive at the campground site. Positive samples were sequenced and found to match DNA sequences from Mesocestoides obtained from island fox and deer mice. Finally, to determine whether ants function as a first intermediate host for Mesocestoides, colony-raised deer mice (n = 47) were fed L. niger (n = 3860) or T. sessile (n = 339) collected from the San Miguel Island fox breeding facility. No mouse became infected with Mesocestoides metacestodes after ingesting ants. While both L. niger and T. sessile from SMI were positive for Mesocestoides DNA, they were not infective to deer mice in the laboratory.     

Taenia taeniaeformis: inactivation of metacestodes by gossypol in vitro

Gossypol, a natural biphenyl compound inhibits Taenia taeniaeformis metacestode development in vivo. In this paper, the direct effect of gossypol on metacestodes was examined. Within 24 hr of incubation at 37 degrees C in greater than or equal to 10(-5) M gossypol, shedding of the tegument from the surface of the metacestodes was observed. There was a significant decrease in [3H]thymidine uptake by T. taeniaeformis in greater than or equal to 10(-5)M gossypol. In addition, NADH lactate dehydrogenase activity of metacestodes was significantly inhibited in greater than or equal to 10(-5) M gossypol. Thus, gossypol has a direct inhibitory effect on T. taeniaeformis metacestodes in vitro.     

Characteristics of the precipitation reaction at the stage of antigen-antibody complex aggregate formation studied by a spectroturbidimetric method]

The possibility of using the spectroturbidimetric method, one of the variants of the light scattering method, for the determination of the characteristics of the second phase of the antigen-antibody reaction was shown examplified by the interaction between rabbit antiserum and bovine serum albumin. The parameters of the aggregates formed in the process (their average size, their concentration by weight and number) were calculated for the whole period of the reaction and with its components taken in different proportions. The precipitation curve plotted by the above method correlated well with the result of an independent determination at an extinction coefficient of E278. A conclusion was drawn, based on special evaluation, that hydrophobic interactions have a predominant influence on the formation of antigen-antibody aggregates. A quantitative approach was proposed for studying the mechanism of the antigen-antibody aggregate formation and for the analytical determination of various classes of immunoglobulins.     

Trypanosoma cruzi: expression of antigens on the membrane surface of parasitized cells

A direct immunofluorescent antibody test with an anti-Trypanosoma cruzi F(ab')2 conjugate was used to demonstrate antigens of T. cruzi on the membrane surface of intact live or fixed macrophages and L929 mouse fibroblasts infected with the organism. Antigens were demonstrated in 5 to 50% of infected cells, and their presence was not directly related to the number of intracellular organisms. Cells with as few as four intracellular amastigotes had demonstrable surface antigens, whereas some cells with as many as twelve or more organisms did not. Capping of antigen-antibody complexes was noted to begin a few minutes after the addition of the anti-T cruzi F(ab')2 conjugate; by 30 min, most of the parasitized cells had eliminated the complexes, and no surface antigen of parasitic nature could be demonstrated. Although capping may have caused a negative result in a previously positive cell, other mechanisms may be involved, because antigens were not demonstrated in some heavily parasitized cells examined immediately after completion of the test. Treatment of the infected cells with trypsin or chymotrypsin resulted in the absence of demonstrable parasite antigens on the cell membrane surface. However, the antigens were again demonstrated 12 hr after the enzymes were removed. The reappearance of parasite antigens on the surface of infected cells was prevented by treatment of the monolayers with puromycin or tunicamycin. A T cell-enriched population of spleen lymphocytes from mice chronically infected with T. cruzi recognized the membrane-bound antigens and proceeded to destroy the host cell and the intracellular organisms. In this process, noninfected cells were also destroyed, possibly because they were coated with antigens released from intact infected cells or from infected cells that had been lysed by the action of the sensitized lymphocytes or their products.