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We undertook a detailed ultrastructural investigation to gain insight into the early stages of development of the vermiform myxozoan, Buddenbrockia plumnatellae Schröder, 1910 in two bryozoan hosts. Early cell complexes arise in the peritoneum after division and migration of isolated cells in the host body wall. The development of cell junctions linking the outer (mural) cells of the complex then produces a sac enclosing a mass of inner cells. Elongation to the vermiform stage (myxoworm) occurs during multiplication and reorganisation of the inner cells as a central core within the single-layered sac wall. The core cells develop into muscle and sporogonic cells separated from the mural cells by a basal lamina. Myogenesis occurs along the length of the myxoworm from cells that differentiate from the central core, and is independent of elongation. Four primary sporogonic cells maintain positions close to the basal lamina, between muscle cells, while giving rise to secondary sporogonic cells that eventually become free in the central cavity. At least some secondary sporogonic cells undergo meiosis. In view of the recent confirmation of the phylogenetic affinity of Buddenbrockia with the Cnidaria, we postulate how features observed in Buddenbrockia may be homologous with cnidarian structures. Finally we propose a new family name, Buddenbrockiidae, to replace Saccosporidae which was proposed previously in breach of the International Code of Zoological Nomenclature.

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.

The microsporidium Trachipleistophora hominis Hollister, Canning, Weidner, Field, Kench et Marriott, 1996, originally isolated from human skeletal muscle cells, inhibited myotube formation from myoblasts when grown in a mouse myoblast cell line C2,C12. Uninfected cultures readily converted to myotubes. Albendazole, a drug with known antimicrosporidial activity, was tested against T. hominis in C2,C12 cells. The drug was added when infection had reached 75% of C2,C12 cells, a level comparable to that obtained in heavily infected muscle in vivo. Doses of 1 ng/ml and 10 ng/ml had no effect on merogony or sporogony. In cultures exposed to 100 ng/ml albendazole, the C2,C12 cells remained in good condition while infection levels dropped to 25% over 7 weeks. Drug doses of 500 ng/ml and 1,000 ng/ml were deleterious to the host cells but some spores retained viability and were able to establish new infections once albendazole pressure was removed. T. hominis meronts exposed to 100 ng/ml albendazole mostly lacked the normally thick surface coat and its reticulate extensions. Meronts were not seen in cultures exposed to higher drug doses. Albendazole at a concentration of 100 ng/ml and higher had a profound effect on spore morphogenesis. There was erratic coiling of the polar tube, often involving the formation of double tubes, and chaotic disposition of membranes which could have been those of polaroplast. The in vitro susceptibility of T. hominis to albendazole was low in comparison with in vitro susceptibility of other microsporidia of human origin.

When in vitro growth of Vittaforma corneae was tested using MDCK, MRC-5, XEN, L-929 and FHM cell lines, propagation occurred only in MDCK, MRC-5 and XEN cells. The intervals required for the various stages of the life cycle to develop were the same in all the cell lines tested. The MDCK cell line was selected to support the growth of V. corneae in vitro and provide the system for in vitro testing of drugs. The weekly output of V. corneae spores from the MDCK cell monolayer was monitored over a 61-week period during which there were fluctuations but no definite increase or decrease in output. Albendazole at 2.1 or 4.2 micrograms/ml in MEM was tested against V. corneae in MDCK cell monolayers and showed antimicrosporidial activity. The percentage of infected cells was reduced in the presence of the drug and there were ultrastructural abnormalities in all stages of the life cycle. The drug prevents parasite division.

Conditions for the effective fluorescence labelling of microsporidian spores by optical brighteners, based on the presence of chitin in the spore wall, are described. Spores of Vairimorpha ephestiae, V. necatrix, V. plodiae, Nosema bombycis, N. apis, N. algerae, Encephalitozoon cuniculi and Enterocytozoon bieneusi were examined. The degree of binding of Calcofluor White M2R (CFW) to untreated spores depends on the conditions and time of storage and the degree of bacterial contamination of the spore sample. Unpurified spores, stored in water are unreliable as control material for the estimation of CFW labelling. However, spores subjected to alkaline treatment by NaOH before CFW application are visualized with ease under all experimental conditions by their bright, not quenching fluorescence in shortwave light (approximately 350 nm) in CFW dilutions of 10(-4) or even lower. Similar improvement in labelling is achieved by exposing spores to CFW dissolved in 0.5-1N NaOH. As well as Calcofluor White M2R other optical brighteners (e.g. Uvitex 2B, Ciba-Geigy or Rylux BA, Ostacolor) can be used for labelling of spores.

A microsporidium isolated in vitro from urine of an AIDS patient was identified as a species of Encephalitozoon. Investigation of the isolate by electron microscopy, amplification of DNA by RAPD PCR, protein analysis by SDS-PAGE and Western blotting and infectivity to athymic mice revealed that it differs from established species of the genus.