The microsporidium Vittaforma corneae (Shadduck, Meccoli, Davis et Font, 1990) develops within the target cell cytoplasm. In the present study, green monkey kidney (E6) cells infected at 30°C, 35°C or 37°C with V. corneae developed enlarged multinucleate structures of up to 200 µm in any horizontal dimension made up either of a single cell or of multiple fused cells. A number of epithelial cell types (SW-480, HT-29, Caco-2 and HCT-8) were infected with V. corneae but did not induce the same highly organized structures, suggesting that for the structure to develop, the host cell must be capable of continued mitosis, and not be differentiated or be detaching from the surface matrix. Live cell imaging of infected E6 cells revealed large, multinucleate infected cells characterized by a central focus from which radiated parasite stages and host cell mitochondria. Immunocytochemistry identifying γ and α tubulin suggested that a single centrally-located microtubule organizing centre governed the distribution of parasite stages and host cell organelles, with mitochondria and parasites being eventually transported towards the periphery of the structure. Whole cell patch clamp analysis of infected cells indicated an average five-fold increase in total membrane capacitance, consistent with an enlarged single cell. Scanning electron microscopy revealed cell-like protrusions around the periphery of the structure with the intervening space being made up of parasites and cell debris. Clearly in the case of V. corneae-infected E6 cells the parasite-host cell relationship involves subverting the host cell cytoskeleton and cell volume control, providing the parasite with the same protected niche as does a xenoma.
Brachiola algerae (Vavra et Undeen, 1970), a parasite of Anopheles mosquitoes, has also been isolated from a human cornea, a cutaneous nodule and deep muscle tissue. All three human isolates of B. algerae are morphologically, serologically, and genetically similar to the mosquito-derived isolates including the original isolate of Vavra and Undeen. All of these isolates grew well in mammalian cell cultures at 37°C and produced spores. Transmission electron microscopy revealed that all developmental stages including meronts, sporoblasts and spores were diplokaryotic and developed in direct contact with the host cell cytoplasm, a feature characteristic of the genus Brachiola. Spores of all isolates reacted well, in the immunofluorescence assay, with the rabbit anti-B. algerae serum. In the immunoblot assay, although the overall banding patterns of the human and mosquito isolates were similar, minor differences could be discerned. Sequencing of the PCR products of the amplified SSU rRNA gene revealed the existence of two distinct genotypes; the original mosquito (Undeen) isolate belonged to genotype 1 and the isolate from cornea and that from the deep muscle biopsy to genotype 2, whereas the isolates from a mosquito and one of the other two human isolates (one from skin abscess) had both genotypes, 1 and 2. It is known that spores of mosquito-derived B. algerae can not only proliferate in mammalian cell cultures at 37°C but also can infect mice when injected into footpads or deposited on the corneal surface. These observations indicate that the spores have potential to be a risk factor for humans, especially those with immunodeficiency.