Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt. 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon. The former is an invasive organism capable of causing life-threatening intestinal and extra-intestinal disease: the latter appears not to be invasive. Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species. However, recent biochemical. immunological, and genetic studies provided convincing evidence that they belong to separate species. Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites. 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra-membranous proteins. 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles. Loose vacuoles containing several bacteria were seen in E. histolytica whereas tight vacuoles containing a single bacterium were observed in E. dispar. Furthermore, bacteria were found only within vacuoles in E. histolytica; in E. dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.     

Evaluation of membrane-bound black bodies in trophozoites and cysts of Naegleria spp

Various Naegleria strains were examined to determine the possible origin and significance of membrane-bound black bodies that were found in all exponentially growing cell populations. The bodies, 40–80 nm in diameter, were distributed randomly in the cytoplasm of Naegleria with ultrastructural features typical of trophozoites. No evidence was obtained that the contents of the black bodies were synthesized in the rough endoplasmic reticulum (ER) and packaged by membranous components, which could be a primitive “Golgi complex” in these amoebae. Examination of cells in various stages of encystment indicated that at least some of the cyst wall material was synthesized and packaged by the rough ER. After condensation into amorphous granules in the cisternae, the cyst wall material appeared in vesicles of the rough ER; these were frequently seen in close proximity to the cell membrane in the vicinity of developing cyst wall. Amorphous granules (～100 nm in diameter), which had variable densities and did not appear to be membrane bound, were seen in the cytoplasm of encysting cells. The substance of these granules also seemed to be incorporated into the cyst wall. The membrane-bound black bodies appeared to be destroyed in lysosomal elements during encystment. The membrane-bound black bodies were concluded to be characteristic of trophozoites and unrelated to encytment of Naegleria.     

Molecular Phylogeny of Marine Gregarine Parasites (Apicomplexa) from Tube‐forming Polychaetes (Sabellariidae,Cirratulidae, and Serpulidae), Including Descriptions of Two New Species of Selenidium

Selenidium is a genus of gregarine parasites that infect the intestines of marine invertebrates and have morphological, ecological, and motility traits inferred to reflect the early evolutionary history of apicomplexans. Because the overall diversity and phylogenetic position(s) of these species remain poorly understood, we performed a species discovery survey of Selenidium from tube‐forming polychaetes. This survey uncovered five different morphotypes of trophozoites (feeding stages) living within the intestines of three different polychaete hosts. We acquired small subunit (SSU) rDNA sequences from single‐cell (trophozoite) isolates, representing all five morphotypes that were also imaged with light and scanning electron microscopy. The combination of molecular, ecological, and morphological data provided evidence for four novel species of Selenidium, two of which were established in this study: Selenidium neosabellariae n. sp. and Selenidium sensimae n. sp. The trophozoites of these species differed from one another in the overall shape of the cell, the specific shape of the posterior end, the number and form of longitudinal striations, the presence/absence of transverse striations, and the position and shape of the nucleus. A fifth morphotype of Selenidium, isolated from the tube worm Dodecaceria concharum, was inferred to have been previously described as Selenidium cf. echinatum, based on general trophozoite morphology and host association. Phylogenetic analyses of the SSU rDNA sequences resulted in a robust clade of Selenidium species collected from tube‐forming polychaetes, consisting of the two new species, the two additional morphotypes, S. cf. echinatum, and four previously described species (Selenidium serpulae, Selenidium boccardiellae, Selenidium idanthyrsae, and Selenidium cf. mesnili). Genetic distances between the SSU rDNA sequences in this clade distinguished closely related and potential cryptic species of Selenidium that were otherwise very similar in trophozoite morphology.     

基于ITS1-5.8S rRNA-ITS2序列的猪源结肠小袋纤毛虫种群特征分析

【目的】为了揭示结肠小袋纤毛虫病在环境中的分子传播机制,研究了猪源结肠小袋纤毛虫的种群特征。【方法】用粪便涂片镜检和改进型DMEM培养基从病猪结肠内容物分离结肠小袋纤毛虫滋养体,然后用显微观察、吖啶橙荧光染色法和基于ITS1-5.8S rRNA-ITS2序列的分子标记技术分析了豫西地区猪群中结肠小袋纤毛虫的种群特征。【结果】结果显示,从来自豫西地区8个县市的病猪中共分离了15株小袋纤毛虫,5.8S rRNA序列相似性高达99.4%以上,同属于结肠小袋纤毛虫,根据ITS1/2序列分析结果,MJ-2和SX-1株属于结肠小袋纤毛虫基因型A,其余13株均属于结肠小袋纤毛虫基因型B。MJ-2和SX-1株滋养体形态特征明显区别其他13株,绝大多数呈球形,运动缓慢,粪便中和体外培养的虫体密度较低;而其他13株的滋养体均呈多形性,运动快速活跃,虫体密度较大。吖啶橙荧光染色显示,2种基因型滋养体的核结构没明显差别。【结论】首次报道中国猪源结肠小袋纤毛虫存在2个基因型,其中基因型B为优势种群,为防控人和动物结肠小袋纤毛虫病提供重要参考。     

Improved Intracellular Morphology of Pneumocystis Carinii From Rat Lung by Postfixation with a Mixture of Potassium Ferrocyanide and Osmium Tetroxide

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO₄ and K₄Fe(CN)₆. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K₄Fe(CN)₆ did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.     

Survival of Giardia lamblia trophozoites after exposure to UV light

The ability of Giardia lamblia trophozoites to reproduce after exposure to different fluences of UV radiation was determined using an in vitro-cultured method. The rate of parasite reproduction following UV exposure was measured by direct enumeration of trophozoites cultured in Diamond's Trypticase Yeast extract-Iron (TYI)-S-33 medium. The results suggested that some G. lamblia trophozoites may survive or are reactivated following exposure to UV fluences up to 10 mJ cm(-2). In addition, trophozoites exposed to a UV fluence of 1 mJ cm(-2) were infectious to Mongolian gerbils. Evidence of survival or reactivation at UV fluences of 20 and 40 mJ cm(-2) was ambiguous and statistically inconclusive, while at 100 mJ cm(-2) there was no evidence of survival or reactivation. This finding may have implications for criteria used by the drinking water and wastewater treatment industry to ensure safe reduction of G. lamblia cysts by UV disinfection processes.     

Conformational Changes in the Cortical Region during Peristaltic Movements of a Gregarine Trophozoite*

SYNOPSIS. Light- and electronmicroscopic studies of noncontracted and contracted regions of Nematocystis magna Schmidt (Gregarinida) trophozoites revealed that their peristalsis-like movements are accompanied by conformational changes in the folded limiting membrane and underlying layer of transverse fibrils. Noncontracted and contracted regions of individual trophozoites had “folded'’and “superfolded'’configurations respectively. Measurements of periodicity and through-focus studies, together with the sequential use of the same Araldite sections for light and electron microscopy, aided in identification of longitudinal fibrils of varying sizes (light microscopy) with specific conformations of the epicytary folds (electron microscopy) in contracted and noncontracted states.     

Fine structure of clonally propagated in vitro life stages of a Perkinsus sp. isolated from the Baltic clam Macoma balthica

We established monoclonal in vitro cultures of a Perkinsus sp. isolated from the baltic clam Macoma balthica and compared morphological features of various life stages by light and transmission electron microscopy to those of the currently accepted Perkinsus species: Perkinsus marinus, Perkinsus olseni, Perkinsus atlanticus, and Perkinsus qugwadi. Except that trophozoites were slightly larger than those of P. marinus, and that they underwent zoosporulation in culture, observation of our isolate under light microscopy did not reveal striking differences from any Perkinsus species. Perkinsus sp. from M. balthica shared fine structural characteristics with other Perkinsus species that clearly place it within this genus. Although zoospores of Perkinsus sp. from M. balthica were slightly smaller than those from other species, the ultrastructural arrangement and appearance of the apical complex and flagella seem to be identical to those of P. marinus and P. atlanticus. Our isolate also appeared, in some sections, to have cortical alveolar expansions of the plasmalemma at regions other than the anterior end and lobulated mitochondria that were reported as unique for P. qugwadi. Little consensus exists among authors in the assignment of taxonomic weight to any particular morphological feature to designate Perkinsus species. The present study of gross morphology and ultrastructure was complemented with molecular studies reported elsewhere, which propose that Perkinsus sp. from Macoma balthica is a distinct species.     

Biochemistry and Metabolism of Giardia

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Pamas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO₂, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphonbosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo. Major lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, sterol (probably cholesterol) and mono-, di- and triacylglycer-ides. The lipid composition of the cysts of G. lamblia isolated from gerbils and G. muris isolated from mice are similar to those obtained from the trophozoites of G. lamblia grown in vitro. The activities of several hydrolases of G. lamblia have been shown to be confined to a single lysosome-like particle population with an equilibrium density of approximately 1.15 in sucrose. Contrary to the trophozoites of Entamoeba and the trichomonads, Giardia trophozoites appear to lack most carbohydrate splitting hydrolases. Calmodulin has been reported in G. lamblia trophozoites, and it appears to have properties similar to the calmodulin isolated from other eucaryotic cells.