The life cycle in vitro of Hymenolepis microstoma (Cestoda).

The cestode Hymenolepis microstoma was grown axenically from oncosphere to infective cysticercoid. Reducing agents were not essential for development and produced structural abnormalities in the organisms. Cysticercoids grown in vitro were excysted, placed in culture, and developed into adults containing gravid segments, thus completing the life cycle in vitro.     

Hymenolepis muris-sylvaticae and H. microstoma: immunological cross-reaction in mice

A primary infection with Hymenolepis microstoma strongly protects against cross-infection with H. muris-sylvaticae and also against secondary infection with H. microstoma in NMRI mice, resulting in an accelerated loss of worms and a weight reduction of the remaining worms. A primary infection with H. muris-sylvaticae causes an accelerated rejection of secondary infection with H. muris-sylvaticae but it has no effect on cross-infection with H. microstoma, neither with regard to worm recovery nor with regard to worm biomass. Determinations by enzyme-linked immunosorbent assay of antibody concentrations in the mouse sera revealed that: (1) the antibody response evoked by H. microstoma infection is much greater than by H. muris-sylvaticae infection; (2) a cross-infection with H. muris-sylvaticae boosts the antibody response evoked by H. microstoma infection; (3) H. microstoma antigen can be used to measure antibody concentration against both H. microstoma and H. muris-sylvaticae; and (4) although H. muris-sylvaticae is rejected faster in a cross-infection (i.e., after a primary H. microstoma infection) than in a secondary infection (i.e., after a primary H. muris-sylvaticae infection), antibodies evoked by the primary H. microstoma infection show little cross-reaction with H. muris-sylvaticae antigen. This suggests that it is doubtful whether serum antibodies are the direct effectors in worm rejection.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Concurrent infections of the trematode Echinostoma caproni and the tapeworms Hymenolepis diminuta and Hymenolepis microstoma in mice

Superimposing the intestinal tapeworm Hymenolepis diminuta on an established infection with the trematode Echinostoma caproni or simultaneous infection of mice with H. diminuta and Hymenolepis microstoma caused destrobilation and expulsion of H. diminuta, whereas establishment and growth of H. microstoma under the same infection regimes were not affected. In contrast, simultaneous superimposition of H. diminuta and H. microstoma on an established E. caproni infection caused destrobilation and expulsion of both H. diminuta and H. microstoma.     

Antagonistic effects of Schistosoma mansoni on superimposed Hymenolepis diminuta and H. microstoma infections in mice

Patent, but not prepatent, Schistosoma mansoni infections in mice enhanced the expulsion of a superimposed infection with Hymenolepis diminuta. An antagonistic effect was also directed against a superimposed H. microstoma infection in mice harbouring patent S. mansoni infections.     

Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos.

The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization. Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir. The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol. After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed. Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages. Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization. Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lumen phase specific cross immunity between Hymenolepis microstoma and H. nana in mice

, and 1988. Lumen phase specific cross immunity between Hymenolepis microstoma and H. nana in mice. International Journal for Parasitology 18: 1019–1027. When mice inoculated with five cysticercoids of Hymenolepis microstoma were challenged with H. nana, they showed strong resistance to challenges with both eggs and cysticercoids of H. nana from day 20. The immunity became complete from day 30 onward: no tissue cysticercoids or lumenal adults of H. nana were established in these mice. However, when mice were challenged with H. nana 10 or 20 days after 10-day old immature H. microstoma were removed by an anthelmintic, the immunity evoked was directed exclusively against the lumenal phase of the cysticercoid challenge but not the tissue cysticercoids of the egg challenge. When mice experienced the prepatent infection with H. microstoma twice, the immunity evoked was also against the lumenal phase of the egg challenge: the oncospheres developed into tissue cysticercoids but thereafter completely failed to develop into lumenal adult tapeworms. Infection with a single cysticercoid of H. microstoma was shown to be sufficient to evoke immunity against H. nana cysticercoid infections in two strains of mice. Sera from mice which experienced a patent infection with H. microstoma revealed that IgG, IgA, IgM isotypes reacted against oncospheres and cysticercoids of both species, while sera from mice which experienced two prepatent infections reacted with cysticercoids only. Sera from H. microstoma infected mice resistant to H. nana caused precipitations on 4-day-old H. nana in vitro. A correlation exists between the presence of stage specific antibodies and resistance to the different stages.     

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)     

Adsorption of bile salts by the cestodes, Hymenolepis diminuta and H. microstoma.

The accumulation of purified sodium taurocholate (NaTC) and sodium glycocholate (NaGC) by Hymenolepis diminuta and Hymenolepis microstoma (Cestoda: Cyclophyllidea) was determined using radioactive bile salts. H. diminuta reached equilibrium levels of approximately 120 nmoles NaTC/g dry wt and 300 nmoles NaGC/g dry wt. Presentation of the bile salts in mixed micelles with 0.35 mM oleic acid did not alter these values. With H. microstoma, the maxima were 195 nmoles NaTC/g dry wt and 614 nmoles NaCG/g dry wt. These values were similarly unaffected by the addition of 0.35 mM oleic acid to the micelles. Equilibrium values of this magnitude, in media containing as much as 25 or 30 mM bile salt, and the maintenance of this level during incubations of 15 to 60 min eliminated the possibility that the accumulation was by diffusion or by any form of mediated transport into the worm. The accumulation on NaTC by H. diminuta was [Na+] independent, and insensitive to ouabain, DNP, and high [K+]. These observations, the maintenance of different levels of NaTC and NaGC, and the failure of the 2 bile salts to compete indicated that there was no active excretion mechanism operating in a fashion similar to the active transport of bile salts in the vertebrate small intestine. It was concluded that the accumulation of NaTC by H. diminuta was actually adsorption to the tegument. Comparable, although more limited, experiments extended this conclusion to the accumulation of NaGC by H. diminuta and of NaTC and NaGC by H. microstoma. It is suggested that bile salt monomers, rather than intact micelles, adsorb to specific loci on the tegument.     

Effect of cysteamine during in vitro maturation on buffalo embryo development

The purpose of this study was to evaluate whether the addition of cysteamine during in vitro maturation (IVM) of buffalo oocytes enhances embryo development. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were matured in vitro in TCM 199 supplemented with 10% fetal calf serum (FCS), 0.5 microg mL(-1) FSH, 5 microg mL(-1) LH, 1 microg mL(-1) 17 beta estradiol and 0 (control), 50, 100 or 200 micromol L(-1) of cysteamine for 24 hours. The matured oocytes then were fertilized and cultured for 7 days. No beneficial effect on maturation and cleavage rate was related to the addition of cysteamine. However, the percentage of embryos that developed to compact morula and blastocyst stage was significantly higher (P < or = 0.01) for oocytes matured in medium containing 50 micromol L(-1) of cysteamine than it was for oocytes matured with 0, 100 and 200 micromol L(-1) cysteamine (22.6% vs 14.9%, 15.7% and 13% respectively); moreover, the addition of 50 micromol L(-1) of cysteamine during IVM significantly (P < or = 0.01) increased the proportion of transferable quality (Grades 1 and 2) embryos (19.3% vs 11.3%, 11.6% and 11.2% respectively). The present study showed that adding a thiol compound (such as cysteamine) to the IVM medium improves buffalo in vitro embryo production (IVEP) efficiency, which so far has been unsatisfactory.