Mature Cysts Research Articles

The ultrastructure of the cyst wall of Giardia lamblia.

Giardiasis is the most common human protozoal infection. In their cystic phase, giardias are protected from the environment by a filamentous cyst wall made up of carbohydrates, proteins, and by two outer membranes separated from the plasma membrane of the parasite by a peripheral space. The present transmission electron microscope observations of G. lamblia cysts of human origin suggest that the extracellular peritrophic space originates from the growth, elongation, and fusion of large cytoplasmic vacuoles. As the large clear vacuoles grew in size, flattening against the inner face of the plasma membrane, they formed a single vacuole that surrounded the body of the parasite, eventually forming two outer membranes. In mature Giardia cysts, the original plasma membrane of the trophozoite becomes the outermost membrane of the cyst wall (CM1). The large vacuoles form a second membrane surrounding the cyst (CM2), and also form a third membrane (CM3), that becomes the new plasma membrane of the trophozoite. During excystation CM1 and CM2 attach to each other and fragment, leaving abundant membrane residues in the peritrophic space. Knowledge of the biochemical composition and functional properties of the complex outer membranous system of G. lamblia cysts here described will be of use to understand the survival of Giardia cysts in the environment, a major factor responsible for the high prevalence of giardiasis worldwide.

Acanthoparyphium tyosenense (Digenea: Echinostomatidae): experimental confirmation of the cercaria and its complete life history in Korea.

Cercaria yamagutii Ito, 1957, was found in the marine mesogastropods Lunatia fortuni and Glossaulax didyma from the tidelands of Simpo located at the estuary of the Mankyoung River, which runs to the western coast of Korea. Metacercariae were found in a marine bivalve Mactra veneriformis after being infected with C. yamagutii experimentally. When a sea gull, Larus crassiostris, was fed with the metacercariae collected from the infected M. veneriformis, adult worms were recovered 10 days later. It was confirmed that the parasites collected from L. crassiostris were Acanthoparyphium tyosenense Yamaguti, 1939. From the results of this life cycle study, it was determined that the first intermediate hosts of A. tyosenense are L. fortuni and G. didyma. The second intermediate and final hosts are M. veneriformis and L. crassiostris, respectively. Mactra veneriformis was experimentally infected with C. yamagutii isolated from L. fortuni and G. didyma by maintaining them in a water tank for 30 min at about 20 C. The cercariae entered M. veneriformis through their incurrent siphons. Five hours after infection, the cercariae tails began to separate from the bodies, and the cercariae formed cysts. Mature cysts were formed 340 hr (14 days) after infection and identified as the metacercariae of A. tyosenense. The prevalence of A. tyosenense metacercariae was 99.5% in naturally infected M. veneriformis. This is the first report of C. yamagutii as the cercaria of A. tyosenense, and the complete life cycle of A. tyosenense was established in Korea.

Giant mature cyst formation following mesh repair of hernias: An underreported complication?

Numerous complications have been described following the implantation of synthetic meshes during hernia repair; one of the rarest, with only three reported cases, is giant mature fibrous cyst formation. Our clinical experience with this complication has led us to believe that it may be more common than previously thought. Surgical operation notes and pathology archives were reviewed for the period January 1998-January 2002 to determine the prevalence of mature cyst formation following mesh repair of hernias. Out of 1100 hernia repair operations involving the use of synthetic meshes in our institution during the period of study, five developed histologically confirmed mature fibrous cysts giving a prevalence of 0.45% for this complication. The formation of a giant mature cyst following mesh repair of hernias is an underreported complication.

Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-beta-hydroxybutyrate synthesis.

The lipids poly-beta-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or beta-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospholipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the enzymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. However, the cysts formed by these strains posses a thicker exine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthesis of alkylresorcinols and encystment even under non-inducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl-CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation.

Apoptosis during spermatogenesis in the spotted ray Torpedo marmorata

This article is a cytological and molecular investigation on the occurrence of apoptosis during spermatogenesis in Torpedo, a cartilaginous fish characterised by a typical cystic testis. Using DNA fragmentation and Bak gene expression, it demonstrated that germ cells undergo apoptosis only at the stages of spermatocyte and spermatid, and degeneration also involves Sertoli but not Leydig cells. In immature cysts, this cellular process probably occurs when the ratio of germ cells to the only Sertoli cell (SC) forming the spermatoblast changes. Apoptosis also takes place in mature cysts after sperm release to eliminate most of the SCs. Few of them, however, become cytoplasts and probably continue secreting androgens so as to control the final events of spermatogenesis, i.e., passage of spermatozoa through the ductus deferentes. Finally, the present investigation demonstrated that, in Torpedo testis, Bak mRNA is expressed during spermatogenesis, thus suggesting that the mitochondrial pathway might be active. This observation in one of the oldest vertebrate classes indicates that, in all vertebrates, the apoptotic process during spermatogenesis is conserved, contributing to testicular homeostasis.

Parasites from farmed ostriches ( Struthio camelus) and rheas ( Rhea americana) in Europe

During a 4-year-period, more than 500 ostriches and several rheas, all born in European countries and raised in Spain and Portugal, have been analyzed for the presence of ectoparasites and endoparasites. A total of 29 parasite species have been found, most of them of the gastrointestinal tract. Some of the helminth species found may represent spureous parasitosis, as only the eggs (of an ascarid and a trematode) were found in some samples. From the organisms identified, the ectoparasites (lice— Struthiolipeurus rheae, S. nandu; mites— Dermoglyphus pachycnemis, Gabucinia bicaudata), helminths (cestoda— Houttuynia struthionis- and nematoda— Libyostrongylus sp., Codiostomum struthionis-) and the ciliate Balantidium struthionis are known as ratite specific parasites. Capillaria eggs and larvae were also found; there are no previous records of this parasite from ostriches, and the data available do not allow to do a temptative specific diagnosis. Among protozoa, most of the species now found are described for the first time in ratites. They include organisms also found in other birds ( Trichomonas gallinae, Tetratrichomonas gallinarum, Chilomastix gallinarum, Spironucleus meleagridis and Pleuromonas jaculans), and organisms whose specific status cannot be established until further analysis are performed ( Cryptosporidium sp., Eimeria sp. and/or Isospora sp., Entamoeba sp. of the one-nucleate and of the eight-nucleate mature cyst groups, Endolimax sp., Iodamoeba sp., Monocercomonas sp., Retortamonas sp., Giardia sp., Blastocystis sp. and euglenids).

Use of recombinant cellulose-binding domains of Trichoderma reesei cellulase as a selective immunocytochemical marker for cellulose in protozoa.

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent beta-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.

Giardia intestinalis glucosamine 6-phosphate isomerase: the key enzyme to encystment appears to be controlled by ubiquitin attachment.

The cyst wall of the parasitic protozoan, Giardia intestinalis, is composed of a polymer of N-acetylgalactosamine, the precursor of which is synthesized by an inducible enzyme pathway. The first enzyme in this pathway, glucosamine 6-phosphate isomerase, is transcriptionally regulated. During encystment and in mature cysts this isomerase appears to be modified by ubiquitin attachment. Thus, it might be targeted for destruction by an ubiquitin-mediated pathway, suggesting that glucosamine 6-phosphate isomerase expression is tightly regulated.

Echinococcosis: A Drop of Water???Review on Human Disease, Diagnosis, and Management

Summary Echinococcosis continues to be a serious public healthproblem throughout the world, especially in sheep-herding areas. Diagnosis should be suspected whenthere is a history of living in, or travel to, an endemicarea in the setting of a characteristic imaging study.Treatment should be tailored for each patient giventheir unique risks and benefits, but most should onlyreceive treatment if symptomatic. Through aggressivevaccination, education, and canine medication, oneday echinococcosis may be as out of date as hydatid isfor a “drop of water.”AcknowledgmentsWe thank Andrew Rhymer for the illustrations and James El-lzy for critical review of the manuscript.

A comparison of cyst age and assay method of the efficacy of contact lens disinfectants againstAcanthamoeba

AIMS(i) To determine effect of Acanthamoeba cyst age, method of production, and (ii) to assay technique on the efficacy of multipurpose solutions (MPS) and hydrogen peroxide based contact lens disinfectants....

A Role for a Galactose Lectin and Its Ligands During Encystment of Entamoeba1

In the life cycle of Entamoeba parasites alternate between the colon-dwelling trophozoite and the infectious cyst forms. The physiologic stimuli that trigger differentiation of trophozoites into cysts remain undefined. On the surface of the human-infecting Entamoeba, parasites express a galactose/N-acetylgatactosamine (gal/galNAc)-binding lectin, which plays demonstrated roles in contact-dependent lysis of target cells and resistance to host complement. Using a reptilian parasite, Entamoeba invadens, to study cyst formation in vitro, we found that efficient encystation was dependent on the presence of gal-terminated ligands in the induction medium. Precise concentration ranges of several gal-terminated ligands, such as asialofetuin, gal-bovine serum albumin (gal-BSA), and mucin, functioned in encystation medium to stimulate differentiation. Greater than 10 mM levels of free gal inhibited the amoeba aggregation that precedes encystation and prevented formation of mature cysts. Inhibitory levels of gal also prevented the up-regulation of genes which normally occurs at 24 h of encystation. The surface of Entamoeba invadens was found to express a gal lectin which has a heterodimeric structure similar to that of Entamoeba histolytica. The 30 kDa light subunit (LGL) of the E. invadens lectin is similar in overall size and sequence to the LGL of E. histolytica. The heavy subunits, however, differ in size, have an identical spacing of cysteines in their extracellular domains, and have highly conserved C-terminal transmembrane and cytoplasmic domains. These results suggest a new role for the Entamoeba gal lectins in monitoring the concentrations of gal ligands in the colon and contributing to stimuli that induce encystment.

Enlarged and astaxanthin-accumulating cyst cells of the green alga Haematococcus pluvialis

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2′,7′-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.

Enlarged and Astaxanthin-Accumulating Cyst Cells of the Green Alga Haematococcus pluvialis

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2',7'-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis.

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2',7'-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10(-3) M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress.

Life history stages of Pyramimonas tychotreta (Prasinophyceae, Chlorophyta), a marine flagellate from the Ross Sea, Antarctica

SUMMARY During a summer cruise to the Ross Sea (Antarctica) areas of snow-covered sea ice were red-coloured due to high concentrations of the recently described Pyramimonas tychotret aDaugbjerg. Light microscopy of living material revealed that the population was comprised of quadriflagellate motile cells and thick-walled cysts. The red colour was due to large numbers of secondary carotenoid-containing granules, positioned in the periphery of motile cells and cysts. Mature cysts also contained numerous starch grains and lipid droplets. Cells from a red-coloured field sample turned green overnight as the secondary carotenoids disappeared when cells were placed in low light conditions. The sample then exhibited the typical grass-green colour of motile cells observed in water samples from the area. Under reduced light motile cells showed strong positive phototaxis. The encystment process involved the asexual transformation of quadriflagellate cells into cysts. A single type of square cyst scale, with perforated floors and walls, replaced the body scales of motile cells. A marked extension, often ending in a hook was at each corner of the cyst scales. Germinating cysts produced four motile cells. Electron microscopy showed the cyst wall to be tri-layered, with a thin, electrondense inner layer, a thick middle layer and a thin outer layer. Sea ice samples with dense populations of motile cells and cyst stages also contained elongate uniflagellate cells. These cells were covered with box scales, foot-print scales, an underlayer of pentagonal scales, limuloid scales and flagellar hair scales identical to those present on the quadriflagellate stage. We tentatively suggest that the uniflagellate stage represents a gamete and its presence implies the occurrence of sexual reproduction. Although, fusion of gametes was not observed, a biflagellate cell with a larger volume was seen which may have been a zygote. How this stage fits into of the life history remains to be explained.

Imaging evaluation of ovarian masses.

Adnexal masses present a special diagnostic challenge, in part because benign adnexal masses greatly outnumber malignant ones. Determination of a degree of suspicion for malignancy is critical and is based largely on imaging appearance. Endovaginal ultrasonography (US) is the most practical modality for assessment of ovarian tumors because it is readily available and has a high negative predictive value. Morphologic analysis of adnexal masses is accurate for identifying masses as either low risk or high risk. The most important morphologic features are non-fatty solid (vascularized) tissue, thick septations, and papillary projections. Color Doppler US helps identify solid, vascularized components in a mass. Spectral Doppler waveform characteristics (eg, resistive index, pulsatility index) correlate well with malignancy but generally add little information to morphologic considerations. Computed tomography can help assess the extent of disease in patients before and after primary cytoreductive surgery. Magnetic resonance (MR) imaging is better reserved for problem solving when US findings are nondiagnostic or equivocal because, although it is more accurate for diagnosis, it is also more expensive. The signal intensity characteristics of ovarian masses make possible a systematic approach to diagnosis. Mature cystic teratomas, cysts, endometriomas, leiomyomas, fibromas, and other lesions can be accurately diagnosed on the basis of T1-weighted, T2-weighted, and fat-saturated T1-weighted MR imaging findings.

Entamoeba sp. (Sarcomastigophora: Endamoebidae) from ostriches ( Struthio camelus) (Aves: Struthionidae)

The first case of Entamoeba of the 1-nucleate mature cyst group in birds is described. Trophozoites and cysts of Entamoeba have been found in ostriches ( Struthio camelus) from farms located all over Spain. The cysts are large (13.47 μm mean diameter); they possess one nucleus when mature, with a large endosome and peripheral chromatine arranged in small granules; chromatoid bodies, when present, are large and elongated. Trophozoites are large (19.88 μm mean diameter), with a clear differentiation between ecto- and endoplasm, this containing numerous vacuoles; the nucleus is large and diffuse. The characteristics of this amoeba resembles but do not completely fulfill those of E. suis and E. chattoni; also, these species are from mammals.

Ultrastructure of a Sarcocystis sp. infecting skinks of the genus Agama.

Macroscopically visible sarcocysts were observed in the skeletal muscles of naturally infected skinks of the genus Agama (infection rate 11.3%). Sarcocysts were described by means of transmission electron microscopy. These cysts measured 0.03-0.25 x 0.38-1.7 mm (mean 0.12 x 1.1 mm). Typical mature cysts were bordered by a primary cyst wall that measured 2.4-5.3 microm (mean 3.9 microm) and was folded into a few nonbranched finger-like protrusions measuring 0.7-1.5 x 1.0-2.5 microm (mean 1.2 x 1.5 microm). These protrusions contained granular elements, but filaments and tubular elements were not observed. A relatively thick, homogeneous tape was observed just underneath the primary cyst wall, measuring 0.5-1.0 microm (mean 0.8 microm) and containing a granulated ground substance in which filaments and tubular elements were not observed. Metrocytes measured 3.1-5.5 x 4.2-7.2 microm (mean 4.0 x 5.8 microm) and merozoites measured 1.2-3.3 x 4.4-8.6microm (mean 2.6 x 7.5 microm). The fine ultrastructural characteristics of both metrocytes and merozoites were similar to those described for many Sarcocystis species and were generally nonspecific.

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

Protective role of astaxanthin against u.v.-B irradiation in the green alga Haematococcus pluvialis

Cyst cells of the green alga Haematococcus pluvialis accumulate astaxanthin with maturation of the resting stage. To study the protective role of astaxanthin against u.v. damage, both immature (astaxanthin-poor) and mature (astaxanthin-rich) cyst cells were exposed to u.v.-A or u.v.-B irradiation, and the residual cell viability and astaxanthin levels were determined. u.v.-B decreased both cell viability and astaxanthin level of cyst cells to a greater extent than u.v.-A. Tolerance of mature cyst cells to u.v.-B was 6-fold higher than that of immature cyst cells. These results indicated that astaxanthin in cyst cells functions as a protective agent against u.v.-B irradiation.