Previous studies have shown that the vacuolar-ATPase (V-ATPase) of the contractile vacuole complexes (CVCs) in Paramecium multimicronucleatum is necessary for fluid segregation and osmoregulation. In the current study, immunofluorescence showed that the development of a new CVC begins with the formation of a new pore around which the collecting canals form. The decorated membranes are then deposited around the newly formed collecting canals. Quick-freeze deep-etch techniques reveal that six 10-nm-wide V-ATPase V₁ sectors, tightly packed into a 20 × 30-nm rectangle, form two rows of these compacted sectors that helically wrap around the cytosolic side of decorated membrane tubules. During new CVC formation, packing of decorated tubules around mature CVCs was temporarily disrupted so that some of these decorated tubules became transformed into decorated vesicles. Freeze-fracturing of these decorated vesicles revealed a highly pitted E-face and a particulate P-face. The V-ATPase was purified for the first time in any ciliated protozoan and shown to contain, as in other cells, the V₁ subunits A to E, and four 14–20 kDa polypeptides. The B subunit was cloned and found to be encoded by one gene containing four short introns. This subunit has 510 amino acid residues with a predicted molecular weight of 56.8 kDa, a value similar to B subunits of other organisms. Except for the N- and C-termini, it has a 75% sequence identity with other B subunits, suggesting that the B subunits in Paramecium, like other species, have been conserved and that the entire surface of this subunit may be important in interacting with other subunits.

Henneguya curimata n. sp. (Myxozoa, Myxobolidae) is described from the kidney of the teleost Curimata inormata collected in an estuarine region of the Amazon River, near Belém, Brazil. This myxosporean produces large cysts (0.6–1.2 mm in diam.) that represent plasmodia containing all life cycle stages, including spores. The spore body is ellipsoidal (∼ 16.6 μm in length and ∼ 6.2 μm in width), and each valve presents a tapering tail (∼ 19.1 μm in length). These valves surround the binucleate sporoplasm cell and two ellipsoidal polar capsules located side-by-side at the same level, measuring 6.5 × 1.2 μm each and containing 10–11 coils of the polar filament. On the basis of its host specificity and on data collected by light and electron microscopy, the organism, H. curimata n. sp. is distinguished as a new species. The taxonomic affinities and morphological comparisons with other similar species of the same genus are discussed.

Nitazoxanide, a 5-nitrothiazolyl derivative, is effective in the treatment of a broad range of parasitic infections. In vitro, it is active against several protozoa, including Cryptosporidium parvum, Blastocystis hominis, and Giardia intestinalis. The objective of this study was to determine the in vitro effect of nitazoxanide on the growth and morphology of three anaerobic protozoa (Entamoeba histolytica, Giardia intestinalis, and Trichomonas vaginalis) and to compare these effects with those of metronidazole and albendazole. A subculture method was used to determine the concentrations required to inhibit growth by 50% or 90% (IC₅₀ and IC₉₀). Nitazoxanide exhibited IC₅₀ and IC₉₀ values of 0.017 and 0.776 μg/ml respectively, against E. histolytica, 0.004 and 0.067 μg/ml against G. intestinalis, and 0.034 and 2.04 6 μg/ml against T. vaginalis. Based on the IC₉₀ values, nitazoxanide was more toxic than metronidazole and albendazole against E. histolytica; albendazole and nitazoxanide were more toxic than metronidazole against G. intestinalis; and metronidazole was the most toxic drug against T. vaginalis. The effects of nitazoxanide on trophozoite ultrastructure of all three parasites included cell swelling and distorted cell shape, a redistribution of vacuoles, plasma membrane damage, and the formation of extensive empty areas in the cytoplasm of the protozoa.

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.

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200—300 nm long, 75–100 nm wide, and have a height of ∼ 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 μm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.

A thermophilic strain of Trimyema minutum was isolated from the hydrothermally heated sea floor at Vulcano Island (Italy) and cultivated monoxenically on Marinobacter sp. and Methanococcus thermolithotrophicus. It can be propagated strictly anaerobically and is sensitive to oxygen: if exposed to air at 48 °C all cells die within 60 min. It grows from 0.45–7.2% (w/v) salt and at pH 6.0–8.0. The isolate is the most extreme thermophilic ciliate which ever has been cultivated, exhibiting an optimal growth temperature of 48 °C (doubling time 6 h). Growth occurs between 28 °C and 52 °C. Trimyema minutum is redescribed using live observation and silver impregnation. Its morphology and the small subunit ribosomal RNA sequence is distinctly different from that of T. compressum, but morphology is highly similar to that of T. shoalsia Nerad et al. 1995, which is thus probably a junior synonym of T. minutum. To stabilize the bewildering species taxonomy in Trimyema, we suggest to recognize our population as a neotype of T. minutum.

Despite being amongst the more familiar groups of heterotrophic flagellates, the evolutionary affinities of oxymonads remain poorly understood. A re-interpretation of the cytoskeleton of the oxymonad Monocercomonoides hausmanni suggests that this organism has a similar ultrastructural organisation to members of the informal assemblage ‘excavate taxa’. The preaxostyle, ‘R1’ root, and ‘R2’ root of M. hausmanni are proposed to be homologous to the right, left, and anterior roots respectively of excavate taxa. The ‘paracrystalline’ portion of the preaxostyle, previously treated as unique to oxymonads, is proposed to be homologous to the I fibre of excavate taxa. Other non-microtubular fibres are identified that have both positional and substructural similarity to the distinctive B and C fibres of excavate taxa. A homologue to the ‘singlet root’, otherwise distinctive for excavate taxa, is also proposed. The preaxostyle and C fibre homologue in Monocercomonoides are most similar to the homologous structures in Trimastix, suggesting a particularly close relationship. This supports and extends recent molecular phylogenetic findings that Trimastix and oxymonads form a clade. We conclude that oxymonads have an excavate ancestry, and that the ‘excavate taxa’ sensu stricto form a paraphyletic assemblage.

Encephalitozoon hellem is a unicellular, obligate intracellular microsporidian species detected and isolated in HIV-infected patients presenting with keratoconjunctivitis, sinusitis, tracheobronchitis, nephritis, cystitis, and disseminated infection. A total of 24 monoclonal antibodies were produced against E. hellem and characterized. The monoclonal antibodies were of the immunoglobulin (Ig) G and Ig M subclasses, and, when incorporated into indirect immunofluorescence and immunoblotting assays, reacted against 13 isolates of E. hellem originating from three geographic regions. These monoclonal antibodies did not react with one strain each of either Encephalitozoon intestinalis or Encephalitozoon cuniculi, demonstrating their specificity. Two monoclonal antibodies reacted with all karyotype B-E. hellem isolates but did not react with karyotype A-isolates from North America and the Netherlands, thus demonstrating antigenic diversity among E. hellem isolates. These results add to the increasing evidence for diversity among E. hellem, which therefore may be reclassified into subspecies.

We have cloned and sequenced a lactate dehydrogenase (LDH) gene from Cryptosporidium parvum (CpLDH1). With this addition, and that of four recently deposited α-proteobacterial malate dehydrogenase (MDH) genes, the phylogenetic relationships among apicomplexan LDH and bacterial MDH were re-examined. Consistent with previous studies, our maximum likelihood (ML) analysis using the quartet-puzzling method divided 105 LDH/MDH enzymes into five clades, and confirmed that mitochondrial MDH is a sister clade to those of γ-proteobacteria, rather than to α-proteobacteria. In addition, a Cryptosporidium parvum MDH (CpMDH1) was identified from the ongoing Cryptosporidium genome project that appears to belong to a distinct clade (III) comprised of 22 sequences from one archaebacterium, numerous eubacteria, and several apicomplexans. Using the ML puzzling test and bootstrapping analysis with protein distance and parsimony methods, the resulting trees not only robustly confirmed the α-proteobacterial relationship of apicomplexan LDH/MDH, but also supported a monophyletic relationship of CpLDH1 with CpMDH1. These data suggest that, unlike most other eukaryotes, the Apicomplexa may be one of the few lineages retaining an α-proteobacterial-type MDH that could have been acquired from an ancestral α-proteobacterium through primary endosymbiosis giving rise to the mitochondria, or through an unknown lateral gene transfer (LGT) event.