Sanitation ability of anaerobic digestion performed at different temperature on sewage sludge.

A small amount of ammonia is used in full-scale plants to partially sanitize sewage sludge, thereby allowing successive biological processes to enable the high biological stability of the organic matter. Nevertheless, ammonia and methane are both produced during the anaerobic digestion (AD) of sludge. This paper describes the evaluation of a lab-scale study on the ability of anaerobic process to sanitize sewage sludge and produce biogas, thus avoiding the addition of ammonia to sanitize sludge. According to both previous work and a state of the art full-scale plant, ammonia was added to a mixture of sewage sludge at a rate so that the pH values after stirring were 8.5, 9 and 9.5. This procedure determined an ammonia addition lower than that generally indicated in the literature. The same sludge was also subjected to an AD process for 60 days under psychrophilic, mesophilic and thermophilic conditions. The levels of fecal coliform, Salmonella spp. helmints ova, pH, total N, ammonia fractions and biogas production were measured at different times during each process. The results obtained suggested that sludge sanitation can be achieved using an AD process; however, the addition of a small amount of ammonia was not effective in sludge sanitation because the buffer ability of the sludge reduced the pH and thus caused ammonia toxicity. Mesophilic and thermophilic AD sanitized better than psychrophilic AD did, but the total free ammonia concentration under the thermophilic condition inhibited biogas production. The mesophilic condition, however, allowed for both sludge sanitation and significant biogas production.

Morphological features of Cloacotaenia megalops (Nitzsch in Creplin, 1829) (Cestoda, Hymenolepididae) from different hosts.

The tapeworm species Cloacotaenia megalops (Nitzsch in Creplin, 1829) is characterized by a very specific morphology. A particularly distinct feature is a large almost square scolex in which four fleshy suction cups are embedded and a rudimentary rostellum. During standard studies of wild duck cestode fauna in northwestern Poland, some morphological differences were observed among specimens, mainly in the shape of scolices and hermaphroditic proglottids of C. megalops isolated from different bird species. This paper attempts to demonstrate the impact of the host species on morphology of the parasite. The study material consisted of 39 individuals of C. megalops (19 from Aythya fuligula, 10 from Anas platyrhynchos, 4 from Bucephala clangula, 4 from Aythya marila and 2 from A. ferina). The isolated parasites were fixed and stored in 70% ethanol and solid preparations were made. Selected external and internal structures of the parasite were measured and photographed using a Zeiss microscope, a microscope camera Opta Tech 2.1 and Opta View 6.0.2.2. It was stated that the sizes of the most important elements of the parasite morphology are generally consistent with those presented in the available literature, nevertheless some differences were observed in both morphology and sizes of some anatomical structures, especially in the form of scolices and hermaphroditic proglottids between specimens isolated from hosts representing three different eco-tribes (Aythyini, Anatini and Mergini).

NO nerves and their targets in a tapeworm: An immunocytochemical study of cGMP in Hymenolepis diminuta.

We studied the pattern of cGMP immunostaining (IS) after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase in adult Hymenolepis diminuta. cGMP-IS was detected in the peripheral nervous system, especially in nerve fibres close to the body muscle fibres. cGMP-IS also occurred in terminals beneath the basal lamina of the tegument and between the muscle fibres of the suckers. The pattern of cGMP-IS was compared to that of 5-HT-IS and GYIRFamide-IS. TRITC-conjugated phalloidin was used to stain the musculature.

The embryonic development of the bodywall and nervous system of the cestode flatworm Hymenolepis diminuta.

Cestodes (tapeworms) are a derived, parasitic clade of the phylum Platyhelminthes (flatworms). The cestode body wall represents an adaptation to its endoparasitic lifestyle. The epidermis forms a non-ciliated syncytium, and both muscular and nervous system are reduced. Morphological differences between cestodes and free-living flatworms become apparent already during early embryogenesis. Cestodes have a complex life cycle that begins with an infectious larva, called the oncosphere. In regard to cell number, cestode oncospheres are among the simplest multicellular organisms, containing in the order of 50-100 cells. As part of our continuing effort to analyze embryonic development in flatworms, we describe here the staining pattern obtained with acTub in embryos and larvae of the cestode Hymenolepis diminuta and, briefly, the monogenean Neoheterocotyle rhinobatidis. In addition, we labeled the embryonic musculature of Hymenolepis with phalloidin. In Hymenolepis embryos, two different cell types that we interpret as neurons and epidermal gland cells express acTub. There exist only two neurons that develop close to the midline at the anterior pole of the embryo. The axons of these two neurons project posteriorly into the center of the oncosphere, where they innervate the complex of muscles that is attached to the hooklets. In addition to neurons, acTub labels a small and invariant set of epidermal gland cells that develop at superficial positions, anteriorly adjacent to the neurons, in the dorsal midline, and around the posteriorly located hooklets. During late stages of embryogenesis they spread and form a complete covering of the embryo. We discuss these data in the broader context of platyhelminth embryology.

A molecular phylogeny of nuclear and mitochondrial sequences in Hymenolepis nana (Cestoda) supports the existence of a cryptic species.

Since isolates of Hymenolepis nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular tools. In the current study, isolates of H. nana from rodent and human hosts from a broad geographical range were sequenced at the ribosomal first internal transcribed spacer (ITS1), the mitochondrial cytochrome c oxidase subunit 1 (C01) gene and the nuclear paramyosin gene loci. Twenty-three isolates of H. nana were sequenced at the ITS1 locus and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences which led to the separation of the isolates into 2 clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Sequencing of a 444 bp fragment of the mitochondrial cytochrome c oxidase 1 gene (C01) in 9 isolates of H. nana from rodents and 6 from humans identified a phylogenetically supported genetic divergence of approximately 5% between some mouse and human isolates. This suggests that H. nana is a species complex, or 'cryptic' species (=morphologically identical yet genetically distinct). A small segment of the nuclear gene, paramyosin, (625 bp or 840 bp) was sequenced in 4 mouse and 3 human isolates of H. nana. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. From the results obtained from faster evolving genes, and the epidemiological evidence, we believe that the life-cycle of H. nana that exists in the north-west of Western Australia is likely to involve mainly 'human to human' transmission.

Morphological and genetic differentiation of Rodentolepis straminea (Goeze, 1752) and Rodentolepis microstoma (Dujardin, 1845) (Hymenolepididae).

The two related species, Rodentolepis straminea (Goeze, 1782) and Rodentolepis microstoma (Dujardin, 1845) (Cestoda, Hymenolepididae), both parasites of rodents, were compared morphologically and electrophoretically. Adult worms were isolated from three wild rodent species of the family Muridae (Apodemus flavicollis, Apodemus sylvaticus, and Mus musculus) from three different sites in Spain and France. Although these two species were strikingly similar in morphological appearance, some of the morphological and metrical features analysed (scolex, mature segments and eggs) can be used for differentiation. Fixed allelic differences were found. Of the ten enzymes detected by starch-gel electrophoresis, six (AAT, AK, GPI, MDH, NP, PGM) showed characteristic isoenzyme profiles in each species. Only in MPI, PEPC, PEPD, and ME enzyme loci were no differences found. The study revealed that the two taxa can be clearly differentiated.

Cytological studies on three hymenolepidid species.

The karyotypes of Vampirolepis nana, Hymenolepis diminuta and Vampirolepis erinacei (Cestoda: Cyclophyllidea) have been described. The number of chromosomes in the diploid cell of the studied taxa was 2n = 12. The absolute and the relative lengths of the chromosomes of V. nana and H. diminuta were measured. The chromosomes had similar metric values. In the karyotypes of V. nana and H. diminuta there were ten one-arm chromosomes and one two-arm chromosome (the third in length in the karyotype of V. nana and the first in length in the karyotype of H. diminuta). The chromosomes in the karyotype of V. erinacei had one arm which was proved by the terminal localization of the constitutive heterochromatin. The examined species differed in the morphology of some chromosomes in the karyotypes.

Cytoskeletal features of the syncytial epidermis of the tapeworm Hymenolepis diminuta.

A hallmark feature of parasitic platyhelminths is a cytoarchitecturally unusual syncytial epidermis composed of a peripheral layer of continuous cytoplasm (the ectocytoplasm) connected to underlying nucleated cell bodies by small cytoplasmic bridges. The helminth epidermis, or tegument, plays important roles in protection and nutrient acquisition; cestodes, in fact, completely lack a gastrointestinal tract and absorb all nutritive material through the tegument. Perhaps not surprisingly, the cestode tegument bears certain resemblances to the mucosal epithelium of the vertebrate small intestine, including the possession of a microvillous brush border upon the surface of the ectocytoplasm. In contrast to the intestinal epithelial cell, however, very little is known concerning the nature and organization of the cytoskeleton within the helminth epidermis. Therefore, a number of different microscopical preparative techniques were used to examine the tegument of the tapeworm Hymenolepis diminuta for the presence and distribution of microfilaments, intermediate filaments, and microtubules. It was found that both actin-containing microfilaments and intermediate-sized filaments are present but are restricted to specific locations along the plasmalemmae of the ectocytoplasm. In contrast, microtubules are found throughout the tegument, and are concentrated in the supranuclear regions of the perikarya and in the cytoplasmic bridges interconnecting the perikarya and ectocytoplasm. Unlike brush borders of most other epithelia, the cestode epidermal brush border lacks a filamentous terminal web and is instead associated with microtubules. A network of fine filaments, 5-8 nm in diameter but distinct from actin-containing microfilaments, runs throughout the ectocytoplasm and appears to interlink tegumental vesicles. These fine filaments may represent the primary "skeletal" system responsible for maintaining the structure of the tegumental cytoplasm.

Developmental changes in Hymenolepis citelli and Hymenolepis diminuta during patency.

Stem cell frequency, wet weight, proglottid number, and egg production were measured in Hymenolepis citelli at specific intervals between 20 and 120 days postinfection in an effort to correlate changes in stem cell frequency to other developmental parameters. Considerable variability was seen in wet weight and proglottid number, but differences did not seem to reflect any relation between these parameters and stem cell frequency. Significant differences were observed in egg production at specific postinfection periods. These appeared to correspond to changes seen in stem cell frequency during patency. Similar changes in egg production which also correspond to measured changes in stem cell frequency were recorded for Hymenolepis diminuta. Differences were also seen in number of eggs contained within gravid proglottids at various times postinfection for both species.

Syncytial epithelia: transport in the absence of paracellular pathways.

The syncytial epithelium of parasitic flatworms offers the opportunity to examine epithelial transport physiology in the absence of paracellular pathways. The asymmetric enzymatic and permeability properties of the apical and basal membranes confirm the transepithelial transport function of the syncytial epithelium. Although the absence of a paracellular pathway has led to the suggestion that the syncytium is a 'tight' epithelium, which would be consistent with its low osmotic and diffusive water permeability, the ion transport mechanisms in the apical membrane are more consistent with those predominating in 'leaky' epithelia. Contrary to that expected of an animal covered with a 'tight' epithelium, the parasitic flatworms are not good ion regulators. The apical membrane contains a Cl-:Na+ co-transport occurs by an active H+ extrusion mechanism, a large part of H+ secretion is coupled to Na+ influx as in 'leaky' will have to await electrical potential profile determinations which are made difficult by the electrical coupling of the syncytium to the underlying nerve-muscle syncytium.

Changes in germinative cell frequencies in the germinative region of Hymenolepis diminuta during development.

Changes in the frequency of germinative cells were measured daily during prepatent development in five-worm infections of Hymenolepis diminuta as well as at regular intervals during patency in five- and 20-worm infections. Frequency changes of this cell were found to correlate closely to the normal growth pattern of this cestode during prepatent development. Significant changes were also seen to occur during patency. However, the pattern of these changes appeared to be modified as the number of worms per host increased. By 90 days postinfection, cestodes recovered from 20-worm infections could be divided into two populations of distinctly different sizes. Significant differences in germinative cell frequency also occurred between these two populations.

Changes in embryonic cell frequencies in the germinative and immature regions of Hymenolepis citelli during development.

This study traces the growth rate of Hymenolepis citelli and correlates such growth to the relative frequency of the only mitotically active cell found in the germinative and immature regions of the cestode. The abundance of this cell, termed the germinative cell, was very high during the early logarithmic growth phase of the cestode and decreased just prior to patency to a level that was maintained until day 90 postinfection. An increase in germinative cell frequency was observed at day 90. It is postulated that this increase may reflect a developmental change which has occurred in the cestode following patency.

The fine structure of spermatogenesis in Hymenolepis diminuta (Cestoda) with a description of the mature spermatozoon.

The processes of spermatogenesis and spermiogenesis in Hymenolepis diminuta were studied by electron microscopy using improved preparative techniques. Spermatogonia (Type A) are characterized by nuclei 3.79 (+/- 0.17) micrometer in diameter, dense cytoplasm packed with free ribosomes and aggregates of mitochondria. After mitoses, certain spermatogonia (Type B) assume syncytial rosettes containing eight nuclei. Primary spermatocytes maintain the rosette syncytium and have large nuclei (4.28 +/- 0.24 micrometer in diameter), smooth endoplasmic reticulum, and polysomes. The secondary spermatocyte is short-lived and is characterized by nuclei (2.0 +/- 0.11 micrometer in diai (2.0 +/- 0.11 micrometer in diameter) and perinuclear membranous lamellae. The syncytial spermatid cluster contains avoid nuclei which condense and elongate to a final diameter of 0.22 +/- 0.04 micrometer. Once elongated, these nuclei become delimited from the syncytium by invaginations of the plasma membrane. During delimitation, cortical peripheral microtubules arise beneath the spermatozoon plasmalemma and a 9 + 1 axoneme extends the length of the mature lance-shaped spermatozoon.

A simple method of obtaining an enriched fraction of tegumental brush border from Hymenolepis diminuta.

A method for isolating an enriched preparation of tegumental brush border from the tapeworm, Hymenolepis diminuta, is described. Combining incubation of whole tapeworms in Krebs-Ringer/tris-maleate solution containing a hemolytic saponin, low shear-force agitation, and differential centrifugation, a pellet is obtained at 2,500 g which contains a significant concentration of surface brush border. The content of brush border in this fraction is identified by the presence of numerous microvilli, increased specific radioactivity after surface tagging with 3H-Concanavalin A, and relatively little mitochondrial contamination (succinic dehydrogenase). Based on morphological criteria, fractions sedimenting with greater force contain dense vesicles and mitochondria from the outer portion of the tegument.

Lipids from subcellular fractions of the tegument of Hymenolepis diminuta.

Lipids comprised 37% and 22.1%, respectively, of the day weights of brush border- and vesicle-rich fractions separated by differential centrifugation of isolated H. diminuta tegument. Neutral lipids of both fractions were rich in cholesterol, but also contained small amounts of glycerides, sterol esters, and (in brush borders) free fatty acids. Phosphatidyl ethanolamine was the most prevalent polar lipid in both fractions, and was particularly abundant (63.4% of total polar lipids) in vesicles; sphingomyelin, not previously reported from H. diminuta, was also present. Polar lipids of both tegumental fractions resembled each other but differed from whole worm polar lipids in fatty acid composition. Tegumental polar lipids contained lower levels of long-chain, polyunsaturated fatty acids than reported for corresponding lipids of whole worms.