Paranucleospora theridion n. gen., n. sp. (Microsporidia, Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis, Copepoda) and Atlantic Salmon (Salmo salar).

Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life-cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle-I) and in the nuclei of epidermal cells (Cycle-II), respectively. Cycle-I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle-II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project.

Brachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity.

The early events of Brachiola algerae (Microsporidia) infection: spore germination, sporoplasm structure, and development within host cells.

Brachiola algerae (Vavra et Undeen, 1970) Lowman, Takvorian et Cali, 2000, originally isolated from a mosquito, has been maintained in rabbit kidney cells at 29 degrees C in our laboratory. This culture system has made it possible to study detailed aspects of its development, including spore activation, polar tube extrusion, and the transfer of the infective sporoplasm. Employing techniques to ultrastructurally process and observe parasite activity in situ without disturbance of the cultures has provided details of the early developmental activities of B. algerae during timed intervals ranging from 5 min to 48 h. Activated and nonactivated spores could be differentiated by morphological changes including the position and arrangement of the polar filament and its internal structure. The majority of spores extruded polar tubes and associated sporoplasms within 5 min post inoculation (p.i.). The multilayered interlaced network (MIN) was present in extracellular sporoplasms and appeared morphologically similar to those observed in germination buffer. Sporoplasms, observed inside host cells were ovoid, contained diplokaryotic nuclei, vesicles reminiscent of the MIN remnants, and their plasmalemma was already electron-dense with the "blister-like" structures, typical of B. algerae. By 15 min p.i., the first indication of parasite cell commitment to division was the presence of chromatin condensation within the diplokaryotic nuclei, cytoplasmic vesicular remnants of the MIN were still present in some parasites, and early signs of appendage formation were present. At 30 min p.i., cell division was observed, appendages became more apparent, and some MIN remnants were still present. By two hours p.i., the appendages became more elaborate and branching, and often connected parasite cells to each other. In addition to multiplication of the organisms, changes in parasite morphology from small oval cells to larger elongated "more typical" parasite cells were observed from 5 h through 36 h p.i. Multiplication of proliferative organisms continued and sporogony was well underway by 48 h p.i., producing sporonts and sporoblasts, but not spores. The observation of early or new infections in cell cultures 12-48 h p.i., suggests that there may also exist a population of spores that do not immediately discharge, but remain viable for some period of time. In addition, phagocytized spores were observed with extruded polar tubes in both the host cytoplasm and the extracellular space, suggesting another means of sporoplasm survival. Finally, extracellular discharged sporoplasms tightly abutted to the host plasmalemma, appeared to be in the process of being incorporated into the host cytoplasm by phagocytosis and/or endocytosis. These observations support the possibility of additional methods of microsporidian entry into host cells and will be discussed.

Microgemma vivaresi (Microsporidia: Tetramicridae): host reaction to xenomas induced in sea scorpions, Taurulus bubalis (Osteichthyes: Cottidae).

Xenomas caused by Microgemma vivaresi Canning, Feist, Longshaw, Okamura, Anderson, Tsuey Tse et Curry, 2005 were found in liver and skeletal muscle of sea scorpions, Taurulus bubalis (Euphrasen). All muscle xenomas examined were in an advanced stage of destruction. In developing xenomas found in liver, parasites were restricted to the centre of the cell, separated from a parasite-free zone by a nuclear network formed by branching of the host cell nucleus. Although xenomas were able to reach a size of several hundred microns, the surface remained a simple plasma membrane. Host reactions took the form of penetration by phagocytes and isolation by fibroblasts. Once the xenoma had been attacked, the nuclear profiles became pycnotic and the barrier between parasitized and parasite-free zones was lost. Parasite antigens cannot be exposed at the surface of intact xenomas, as the host does not recognise the enlarging cell as foreign. Breaches in the plasma membrane of the xenoma and leakage of parasite antigens are thought to be the stimuli for phagocyte entry into the cell, its isolation by fibroblasts and eventual granuloma formation.

In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish.

Anncaliia algerae is an aquatic microsporidium that most commonly infects mosquitoes but can be grown on the rabbit kidney cell line, RK-13. Spores were purified from RK-13 cultures and added to cell lines from warm water fish and from an insect. The cell lines were GFSK-S1 and GFB3C-W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27°C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish, and insects.

Transfer of the members of the genus Brachiola (microsporidia) to the genus Anncaliia based on ultrastructural and molecular data.

Two microsporidian genera, AnncaliiaIssi, Krylova, & Nicolaeva 1993 and BrachiolaCali et al. 1998, possess a Nosema-type life cycle and unique cell surface ornamentations, which include precocious electron-dense coating of the plasmalemma and a variety of secretory structures deposited on the parasite surface and scattered in the host cell cytoplasm. Comparative analysis of ultrastructure of Anncaliia meligethi (the type species of the genus Anncaliia) and of B. vesicularum and B. algerae (the best-studied members of the genus Brachiola) clearly demonstrated that these microsporidia share many distinctive morphological features. The comparison of small subunit ribosomal DNA sequences showed high sequence identity of A. meligethi and B. algerae. Phylogenetic analyses indicated that the rDNA sequences of A. meligethi clustered with those of B. algerae suggesting a close relatedness of these microsporidia. The combination of molecular and morphological data provided clear evidence that these microsporidia belong to the same genus and therefore, warranted emendation of the genus Anncaliia and establishments of the following new combinations: Anncaliia vesicularum nov. comb., Anncaliia algerae nov. comb., Anncaliia connori nov. comb., and Anncaliia gambiae nov. comb. The generic name Brachiola is submerged according to the rule of priority.

Transmission and serial propagation of Enterocytozoon bieneusi from humans and Rhesus macaques in gnotobiotic piglets.

For over a decade Enterocytozoon bieneusi infections in people with AIDS have been linked with chronic diarrhea and wasting. The slow scientific progress in treating these infections is attributed to the inability of investigators to cultivate the parasite, which has also precluded evaluation of effective therapies. We report here successful serial transmissions of E. bieneusi from patients with AIDS and from macaques with AIDS to immunosuppressed gnotobiotic piglets. One infected piglet was still excreting spores at necropsy 50 days after an oral challenge. Spores in feces were detected microscopically by trichrome stain and by PCR and within enterocytes by in situ hybridization and immunohistochemistry. E. bieneusi infection induced no symptoms. The development of an animal model for E. bieneusi will open up new opportunities for investigating this parasite.