Monohydrocalcite in calcareous corpuscles of Mesocestoides corti

Mesocestoides corti (syn. vogae), as many other cestode platyhelminthes, contains abundant mineralized structures called calcareous corpuscles. These concretions may constitute as much as 40% of the dry weight of the organisms, but their function remains poorly understood. In this work, we reviewed the mineral composition of the calcareous corpuscles of M. corti. X-ray diffraction pattern showed that the major mineral component of the corpuscles is a hydrated form of calcium carbonate, monohydrocalcite, also confirmed by infrared spectrometry. The baseline shift of the X-ray diffraction spectra suggested the presence of amorphous calcium carbonate, accordingly to previous reports, and an organic matrix was confirmed by FTIR. Monohydrocalcite is a rare mineral unusually found in biominerals. Although the significance of monohydrocalcite in biominerals has not been determined, the knowledge of corpuscles composition is of relevance to establish their function and for the elucidation of the mechanisms involved in mineralization processes.     

Environmental temperature and the growth of Mesocestoides corti populations in mice

The volumes of tetrathyridial populations in the peritoneal cavities of mice of both sexes kept at 5 ± 1dgC for 20 days were significantly larger than those in control mice kept at 21 ± 1dgC; the volumes of these populations in mice kept at 35 ± 1dgC were significantly lower in males but not in females. The liver weights in different groups were proportional to the intensity of liver infection: for both sexes livers were heaviest at 5 ± 1dgC and lightest at 35 ± 1dgC.     

Some enzymes of carbohydrate metabolism in Mesocestoides corti and Heterakis spumosa

The activities of selected enzymes of carbohydrate metabolism were measured in tetrathyridia of Mesocestoides corti and in adult females and males of Heterakis spumosa. When the species were compared, only lactate dehydrogenase and phosphoenolpyruvate carboxykinase activities were considerably higher in M. corti. Activities of other enzymes were higher in H. spumosa, with malate dehydrogenase activity being considerably so. In H. spumosa, enzyme activity was higher, and succinate dehydrogenase markedly so in males, when compared with females. Tetrathyridia aged 170 and 210 days show relatively stable malate and lactate dehydrogenase activities, and mice of ICR and BALB/c strains are suitable for the maintenance of tetrathyridia.     

Pathological changes in mice infected with tetrathyridia of Mesocestoides corti.

Long-term studies in mice experimentally infected with the tetrathyridia of Mesocestoides corti have revealed disseminated lesions in the liver, lung, kidney, epididymis, and testicle.     

Asexual multiplication of tetrathyridia of Mesocestoides corti in Crotalus viridis viridis

Prairie rattlesnakes, Crotalus viridis viridis Rafinesque, 1818, were infected with tetrathyridia of Mesocestoides corti Hoeppli, 1925, by means of oral intubation. Snakes were maintained at constant temperatures of either 25, 30, or 35 C for 4-24 wk. Postmortem examinations revealed the first experimental evidence of asexual multiplication of tetrathyridia in a laboratory-infected ectothermic host. Time and temperature were statistically significant with P values of less than 0.05.     

Effect of praziquantel on the strobilar development of Mesocestoides corti in vitro

The effect of praziquantel (PZQ) on the strobilar development of the cyclophyllidean cestode Mesocestoides corti was explored. Mesocestoides corti larvae were cultivated under conditions reported to favour their differentiation to the adult stage. Parasites were exposed to 0.1 microg ml(-1) PZQ for 16 h and subsequently transferred to drug-free medium. The ocurrence of segmentation--an early event of the larval somatic differentiation to the adult worm-- was considered as quantitative data. This phenomenon was evidenced earlier in worms transiently exposed to PZQ with respect to control cultures. Moreover, the rate of segmentation of drug-treated worms at the end of the experiment almost doubled that of control worms. To date, no similar effect on any cestode developmental process has been reported for an anthelmintic drug. In the light of the existing knowledge and understanding of PZQ mechanisms of action, the proposed experimental approach could contribute to the elucidation of pathways and mechanisms involved in cestode strobilar development.     

In vitro segmentation induction of Mesocestoides corti (Cestoda) tetrathyridia

Mesocestoides corti is a suitable model for studying cestode development because of its ability to reproduce asexually and segment in vitro. The cultured parasite is also capable of sexual differentiation and, probably, reproduction. To establish conditions that increase the efficiency of in vitro M. corti larvae (tetrathyridia) segmentation, we tested the effects of an inducing agent and some physical parameters in cultures. We found that a 5% CO2-95% N2 gas phase, an incubation temperature of 39 C (instead of 37 C), and a 24-hr pretreatment with trypsin (10(5) BAEE/ml, BAEE = Na-benzoil-L-arginine ethyl ester unit of trypsin activity) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 20% fetal bovine serum (FBS) are able to increase individually or synergistically the segmentation rate of tetrathyridia. A segmentation rate of up to 100% was achieved on day 4 of culture, when all these conditions were used simultaneously, in comparison with an average rate of 40% obtained not before day 11 in cultures without any inducing treatment. Fetal bovine serum is essential for segmentation, and a concentration of 20% was established as the standard for induction.     

Anaerobic and aerobic energy metabolism in the larvae (tetrathyridia) of Mesocestoides corti

The tetrathyridia of Mesocestoides corti produce lactate, succinate, acetate, and CO₂ as major carbon-containing end products during in vitro incubation with glucose as the substrate. Differences in the rate of glucose consumption and lactate production under anaerobic or aerobic conditions were observed, but their significance could not be determined. However, succinate production was greatly decreased in the presence of oxygen.The relative activities and intracellular distribution of various enzymes involved in energy-supplying metabolism of the larvae appear to conform to the pathways observed in other parasitic helminths known to produce lactate, succinate, and volatile fatty acids as metabolic end products. Some common features found in this respect are the relatively low pyruvate kinase activity, the presence of a highly active cytoplasmic phosphoenolpyruvate carboxylase and the capability of mitochondrial membrane bound fumarate reductase to reduce fumarate by means of NADH. Although a stimulatory effect of fructose-1,6-diphosphate on the reaction velocity of pyruvate kinase occurred, the absolute activity of this enzyme is very low.Nearly all the enzymes required for Krebs cycle activity are available in the tetrathyridia. Under the assay conditions employed by us, only NAD-dependent isocitrate dehydrogenase could not be demonstrated. The small amounts of ¹⁴CO₂ liberated from 6-¹⁴C-glucose suggest that the cycle in its classical form probably only functions at a very low rate. The incorporation of ¹⁴C from labeled glucose into glycogen indicates the presence of enzymes capable of glycogenesis. The incorporation rate was found to be higher in the presence of oxygen than under anaerobic conditions. On account of the very low NAD-linked glycerol-3-phosphate dehydrogenase activity the glycerolphosphate cycle may be of minor importance for the tetrathyridia.As a result of these studies a scheme for the main carbohydrate dissimilating pathways in the tetrathyridia is proposed and the significance of oxygen with respect to energy-supplying metabolism is discussed.     

Maternal transmission of asexually proliferative Mesocestoides corti tetrathyridia (Cestoda) in mice

Asexually proliferative Mesocestoides corti tetrathyridia were studied to test the hypothesis of in utero transmission in mice and define more clearly the path of transmammary transmission. In utero transmission was not observed in 132 fetuses (22 litters) taken by caesarean section from infected mothers. However, 19 of these mothers had tetrathyridia in their mammary glands at the time of operation, nine had worms in the uterine lumen, and one had a single worm in the maternal blood space of a placenta. No tetrathyridia were found in amniotic cavities. No infection was found in 32 young (7 litters) examined immediately after birth to infected mothers, but before nursing. No infection was found in 30 young (5 litters) removed from infected mothers before nursing and raised by uninfected fosters. Of 29 uninfected young (5 litters) allowed to nurse on infected mothers, 18 became infected. Whole mounts and sections of infected mammary glands showed proliferating tetrathyridia free in larger milk ducts and free and encapsulated in mammary parenchyma. These data suggest that maternal transmission of M. corti tetrathyridia in mice occurs primarily or perhaps exclusively by the transmammary route.     

Mesocestoides corti (syn. vogae, cestoda): characterization of genes encoding cysteine-rich secreted proteins (CRISP)

With the aim of identifying genes involved in development and parasite adaptation in cestodes, four coding sequences were isolated from the cyclophyllidean Mesocestoides corti larval stage (tetrathyridium). Genes showed significant similarity to the cysteine-rich secreted protein (CRISP) encoding genes, a large family that includes stage and tissue-specific genes from diverse organisms, many associated with crucial biological processes. The full-length McCrisp2 cDNA encodes a predicted protein of 202 residues in length, containing 10 cysteines and a putative signal peptide. The expression level of McCrisp2 was estimated by Real-time PCR, relative to GAPDH, showing an increase of 75% in segmented worms compared to tetrathyridia. By in situ hybridization, McCrisp2 expression was localized mainly at the larvae apical region of tetrathyridia and in the proglottids of segmented worms. Taken together our results suggest a possible role for M. corti CRISP proteins as ES products, potentially involved in differentiation processes as proposed for homologs in other organisms.     

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infections in mice.

The influence of Mesocestoides corti on subsequent Angiostrongylus cantonensis infection in mice (C57BL/6 and BALB/c) was assessed. Both strains of mice infected with M. corti demonstrated a peak blood eosinophilia at around 3 weeks post-infection (p.i.). C57BL/6 and BALB/c mice primarily infected with M. corti were given A. cantonensis infection 18 days later, but pre-existing M. corti infection did not affect the recovery of intracranial worms of A. cantonensis at day 21 p.i. BALB/c mice with mixed parasite infections showed low morbidity and mortality as compared with mice singly infected with A. cantonensis and some mice demonstrated a pulmonary migration of intracranial worms. In C57BL/6 mice, intracranial worms were killed and thus all mice survived. C57BL/6 mice with mixed parasite infections failed to resist A. cantonensis reinfection. The blastogenic responses of spleen cells against A. cantonensis antigen were lower in BALB/c than in C57BL/6 mice and mixed parasite infections also resulted in less blastogenic responses against both concanavalin A and A. cantonensis antigen than monoinfection. The recovery of M. corti biomass was significantly higher in mice with mixed parasite infections than mice with monoinfection with M. corti. These data suggest a distinct difference in response to A. cantonensis infection between C57BL/6 and BALB/c mice, and the induction of immunosuppression in both mouse strains following M. corti infection. Blood eosinophilia provoked by M. corti infection is not directly associated with the killing of worms in subsequent A. cantonensis infection.     

Circulating antigens in infections of mice by tetrathyridia of Mesocestoides corti Hoeppli, 1925

Two circulating antigens were detected in the serum of ICR/Timco female mice infected intraperitonealy with tetrathyridia of the cestode Mesocestoides corti Hoeppli, 1925. One circulating antigen appeared by day 2 postinfection (p.i.) and remained in all mice until at least 90 days p.i. A second antigen appeared in the serum on day 14 p.i. and disappeared from all mice by day 28 p.i. Infected mouse serum also contained antibodies against one secretory/excretory antigen and two antigens in crude homogenate, as judged by double diffusion in two dimensions (Ouchterlony). Immune deposits were observed in the kidney tissue of Rockland mice by transmission electron microscopy, and their identity as products of tetrathyridia was confirmed by immunofluorescence. Further studies showed that the main antibody subclass associated with the mesangial immune deposits was 7S gamma l, and that other subclasses of IgG and IgM were not involved. Antigen was found in the proximal renal tubules of infected mice, as demonstrated by fluorescein-labeled IgG fraction of rabbit antitetrathyridia secretory/excretory antigen antisera. The presence of tetrathyridia antigen in the urine of infected mice was confirmed using the Ouchterlony technique.     

Factors influencing the establishment of Mesocestoides corti in mice following oral inoculation of tetrathyridia

Factors which influence the establishment of tetrathyridia of Mesocestoides corti in mice following inoculation per os were examined. Only a proportion of the tetrathyridia penetrate the gut wall and gain access to the peritoneal cavity and liver, and most of these penetrate through the wall of the small intestine. It appears that tetrathyridia must attach to the intestinal mucosa and commence penetration immediately or they pass into the large intestine and are voided. Establishment was not influenced by strain, sex or age of host. However, the temperature at which tetrathyridia were maintained before inoculation influence their ability to penetrate the intestinal wall. Additionally it appears that tetrathyridia have to undergo a morphological change before or during, this penetration phase.     

In vitro culture of tetrathyridia of Mesocestoides corti using a gel based diphasic medium

Tetrathyridia of Mesocestoides corti were cultured in vitro in a diphasic medium consisting of a liquid medium (CMRL Sigma) and a thixotropic nutrient gel (Oxoid). Tests demonstrated that a 50% medium/gel mixture produced optimum conditions for the survival and development of tetrathyridia. Established anthelminthic drugs were inoculated into the gel which demonstrated that this system can be used for preliminary anthelminthic drug screening. The development and survival of the tetrathyridia were influenced by the addition of pepsin, trypsin and liver peptone to the culture media. The development and maturation of proglottids were observed in addition to asexual reproduction by the process of budding. Tetrathyridia maintained in vitro and reinfected into both mouse and rat hosts retained their viability.