A real-time PCR method for quantifying viable ascaris eggs using the first internally transcribed spacer region of ribosomal DNA

Worldwide, 1.4 billion people are infected with the intestinal worm Ascaris lumbricoides. As a result, Ascaris eggs are commonly found in wastewater and sludges. The current microscopy method for detecting viable Ascaris eggs is time- and labor-intensive. The goal of this study was to develop a real-time quantitative PCR (qPCR) method to determine the levels of total and viable Ascaris eggs in laboratory solutions using the first internally transcribed spacer (ITS-1) region of ribosomal DNA (rDNA) and rRNA. ITS-1 rDNA levels were proportional to Ascaris egg cell numbers, increasing as eggs developed from single cells to mature larvae and ultimately reaching a constant level per egg. Treatments causing >99% inactivation (high heat, moderate heat, ammonia, and UV) eliminated this increase in ITS-1 rDNA levels and caused decreases that were dependent on the treatment type. By taking advantage of this difference in ITS-1 rDNA level between viable, larvated eggs and inactivated, single-celled eggs, qPCR results were used to develop inactivation profiles for the different treatments. No statistical difference from the standard microscopy method was found in 75% of the samples (12 of 16). ITS-1 rRNA was detected only in samples containing viable eggs, but the levels were more variable than rDNA levels and ITS-1 rRNA could not be used for quantification. The detection limit of the rDNA-based method was approximately one larvated egg or 90 single-celled eggs; the detection limit for the rRNA-based method was several orders of magnitude higher. The rDNA qPCR method is promising for both research and regulatory applications.     

On the oesophagus of ascaris lumbricoides

Summary The oesophagus ofAscaris lumbricoides is built up by a muscular-epithelial part containing the ordinary muscle fibres which run from the three sides of the oesophagus lumen towards the periphery and a system of fibres and fibrous plates at the margin of the triradiate oesophagus lumen. In four complete series ofA. lumbricoides (males and females) the nuclei of both kinds of fibres were constant in arrangement and number. The glandular system of the oesophagus consists of a large dorsal gland and two smaller subventral ones. The opening of the dorsal gland is at the anterior end of the oesophagus in the middle between nerve ring and lips. The openings of the two subventral glands are placed in the posterior end of the oesophagus in the middle of the two subventral slides at the same level. The dorsal gland fills with its ramifications the space between the muscle fibres of the three sectors with the exception of the most anterior end of the oesophagus before the opening of the dorsal gland and the subventral sectors in the posterior end of the oesophagus. The subventral sectors of the posterior end are filled by the ramifications of the two subventral glands. The ramifications of these three glands are connected. The three nuclei of the three glands have their place in the posterior end of the oesophagus, the nuclei of the subventral glands are situated in the most ventral part of each subventral sector close to each other, the nucleus of the dorsal gland has a complicated form and extends over the whole breadth of the dorsal sector. With 10 figures in the text.     

Demonstration and possible function of NADH:NAD+ transhydrogenase from ascaris muscle mitochondria.

Mitochondria from the muscle of Ascaris lumbricoides var. suis function anaerobically. NADH is generated in the intermembrane space as a consequence of the "malic" enzyme reaction. It has been suggested that this reducing equivalent in the form of hydride ion, would be translocated across the inner membrane in order to mediate ATP generation via the fumarate reductase reaction. In accord with this suggestion, intact Ascaris mitochondria showed appreciable NADH oxidase activity. Sonication resulted in an approximately 2-fold increase in NADH oxidase activity, whereas "malic" enzyme, fumarase, and NADH:NAD+ transhydrogenase activities increased approximately 7- to 14-fold, respectively. Phosphorylation capabilities and permeability toward pyridine nucleotides also indicated the intactness of the mitochondria. Ascaris mitochondria incubated anaerobically in the presence of fumarate, and [14C]NADH catalyzed a rapid reduction of the fumarate to succinate with the concomitant formation of equivalent quantities of extramitochondrial NAD+. However, very little isotope was recovered from the washed mitochondria, indicating the possibility of hydride ion translocation in the absence of nucleotide translocation. NADH:NAD+ transhydrogenase has been isolated from the muscle mitochondria of the intestinal nematode, Ascaris lumbricoides var. suis. The enzyme seems to have been solubilized from the mitochondrial membrane fraction by treatment with sodium deoxycholate followed by dialysis and subsequent adsorption by and elution from alumina C gamma. No NADPH:NAD+ transhydrogenase activity was detectable, making the Ascaris system unique over others reported. Activity was protected by L-cysteine, reduced glutathione and dithioerythritol, but strongly inhibited by low concentrations of p-chloromercuribenzoate or silver nitrate. The thionicotinamide derivative of NAD+ (thioNAD+) was employed to accept hydride ions from NADH in order to assay spectrophotometrically at 398 nm. Apparent Km values for thioNAD+ and NADH were 1 X 10(-4) M and 8 X 10(-6) M, respectively. That the physiological nucleotide, could act as hydride ion acceptor from NADH was indicated by the findings that NAD+ competitively inhibited the reduction of thioNAD+ when assayed at 398 nm. The additional finding of a noncompetitive inhibition between NAD+ and NADH suggested at least two binding sites on the enzyme, one for NADH and another common site for NAD+ and thioNAD+. More conclusive evidence indicating the participation of NAD+ as acceptor was obtained by incubation of the enzyme with NADH and [14C]NAD+ and demonstrating a rapid formation of [14C]NADH. These findings, in conjunction with those discussed above, suggest a physiological function of this enzyme in hydride ion translocation.     

The cytochemistry of Limulus eggs.

Cytochemical studies on uninseminated mature eggs of Limulus demonstrate the presence of carbohydrates, lipids and proteins in the egg envelopes and yolk. The vitelline envelope, cortical region and yolk are rich in 1,2-glycols, with the vitelline envelope, containing fewer reactive 1,2-glycol groups than other components of the egg. Neutral mucopolysaccharides are found in the cortical region and yolk, but only the cortical region of the eggs demonstrate the presence of sulfated mucosubstances (which are in part glycoprotein in nature) and glucose-6-phosphatase. Protein is evident in all egg components. Biochemical analysis demonstrate the protein in the egg envelopes of uninseminated eggs is composed of sixteen amino acids while that of developing eggs contain seventeen amino acid residues. Electrovalent linkages and non-S-S- covalent linkages between protein chains are shown to be instrumental in maintaining the stuctural integrity of Limulus egg envelopes. Neutral lipids, unsaturated lipids, phospholipids and fatty acids are demonstrated in yolk bodies and lipoproteins, unsaturated lipids and fatty acids constitute part of the egg envelopes. DNA is concentrated in the cortical region and the yolk bodies     

Non-surgical recovery of bovine eggs.

An improved, non-surgical technique for recovering bovine eggs is described using modified Foley catheters. Egg recovery rates per attempt were, $\text{36}\text{51}$ (71%), for normal, unsuperovulated donors and $\text{4}\text{38}$ (11%), for unsuperovulated donors with known fertility problems. For superovulated donors, eggs were collected in $\text{24}\text{26}$ attempts (92%) averaging 6.9 per recovery.     

Experimental activation of ascidian eggs.

The effects of the ionophore A23187 on the activation of the eggs of Ascidia malaca have been studied. No common external ion in the sea water is found to be essential for the activation but lanthanum and manganese inhibit the response. These observations support the interpretation that activation of these eggs results from changes in free intracellular calcium levels. This has led to the prediction of two other activating treatments, namely high external calcium and addition of theophylline.     

Hatching of Daphnia sexual eggs. I. Intraspecific differences in the hatching responses of D. magna eggs

• 1 To test the hypothesis that the variability in hatching response of the sexual eggs of Daphnia has a genetic component, hatching after a standardized decapsulation technique was studied in different D. magna families, resulting from intra- as well as interclonal crosses.
• 2 There were significant differences in hatching response between families. Average hatching rates ranged from 0.0% to 81.9%, depending on the family under study.
• 3 Offspring-on-parent regressions indicate that the hatching rate-of sexual eggs is to a large extent determined by the genotype of the mother (maternal inheritance).
• 4 Our results suggest that there is ample generic variation on a microgeographic scale for characteristics related to hatching of sexual eggs in Daphnia.

     

Chloride permeability of sea urchin eggs.

We have examined the content and permeability of chloride in sea urchin eggs. After fertilization there is a large increase in the permeability to chloride. We discuss the mechanism underlying this permeability change and the generalized increase in ion permeability observed after fertilization.