Small subunit ribosomal DNA phylogeny of microsporidia that infect Daphnia (Crustacea: Cladocera).

Glugoides intestinalis, Microsporidium sp., Ordospora colligata, Gurleya vavrai, Larssonia obtusa and Flabelliforma magnivora are microsporidian parasites of planctonic freshwater crustaceans Daphnia spp. We performed a phylogenetic analysis of the small subunit ribosomal DNA which revealed their positions as polyphyletic. G. intestinalis, Microsporidium sp. and O. colligata, which are horizontally transmitted gut parasites with small spores and low virulence, group with different lineages. G. intestinalis is related to 2 microsporidia infecting lepidopterans and to Vittaforma corneae, which has been described as a human pathogen. It is thought that V. corneae may have an invertebrate as its natural host. Microsporidium sp. is a relative of the genera Enterocytozoon and Nucleospora, pathogens of man and fish respectively. O. colligata is the first species found to be closely related to the genus Encephalitozoon, which is comprised of 3 species that are parasites of homeothermic vertebrates. G. vavrai and L. obtusa are sister taxa that branch close to the Amblyosporidae, the only microsporidia with known intermediate hosts. This finding supports the presumption of secondary hosts for G. vavrai and L. obtusa, as it has not been possible to maintain these species in Daphnia in the laboratory. F. magnivora roots deep at the base of the phylum microsporidia with no close relative found so far.

Growth of Trachipleistophora hominis (Microsporidia: Pleistophoridae) in C2,C12 mouse myoblast cells and response to treatment with albendazole.

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.

Serological differentiation of microsporidia with special reference to Trachipleistophora hominis.

Myositis is a common clinical syndrome in advanced stages of AIDS. Trachipleistophora hominis (phylum Microspora) has been detected in several cases of painful, immobilising myositis in AIDS patients. Enzyme linked immunosorbent assays (ELISAs) and Western blotting of protein profiles separated by SDS PAGE were used to determine whether this species could be detected and differentiated by serology. Sixteen microsporidia, including several species known to infect man and species infecting fish, crustaceans and a mosquito, were used as antigen. Each species had a unique profile of SDS PAGE-separated proteins. In Western blots, mouse antiserum, raised to T. hominis and selected for its high ELISA specificity, bound to antigens ranging from less than 25 kDa to greater than 250 kDa with major bands at 39-44 kDa and 98-150 kDa on T. hominis protein profiles. The serum also recognised some high molecular weight antigens in the profiles of Vavraia culicis, Heterosporis anguillarum, and three species of Pleistophora but none in the remaining genera examined. It was concluded that ELISA and Western blotting could be used to detect and differentiate T. hominis in muscle biopsy tissue from patients with myositis. However, sera from T. hominis-infected patients in the terminal stages of AIDS would not be useful for detection of infections because of a sharp decline in antibody level.

Relationships of microsporidian genera, with emphasis on the polysporous genera, revealed by sequences of the largest subunit of RNA polymerase II (RPB1).

Molecular data have proved useful as an alternative to morphological data in showing the relationships of genera within the phylum Microsporidia, but until now have been available only for ribosomal genes. In previous studies protein-coding genes of microsporidia have been used only to assess their position in the evolution of eukaryotes. For the first time we report on the use of a protein-coding gene, the A-G region of the largest subunit of RNA polymerase II (RPB1) from 14 mainly polysporous species, to generate an alternative phylogeny for microsporidia. Using the amino acid sequences, the genera and species fell into the same main groupings as had been obtained with 16S rDNA sequences, but the RPB1 data provided better resolution within these groups. The results supported the pairings of Trachipleistophora hominis with Vavraia culicis and Pleistophora hippoglossoideos with Pleistophora typicalis. They also confirmed that the genus Pleistophora is not monophyletic and that it will be necessary to transfer Pleistophora ovariae and Pleistophora mirandellae into one or more other genera, as has already been effected for Pleistophora anguillarum.

Phylogenetic relationships of Pleistophora-like microsporidia based on small subunit ribosomal DNA sequences and implications for the source of trachipleistophora hominis infections.

The microsporidian Trachipleistophora hominis was isolated in vitro from the skeletal muscle of an AIDS patient. Since its discovery several more cases of myositis due to Trachipleistophora have been diagnosed but the source of infection is unknown. Morphologically, T. hominis most closely resembles Pleistophora and Vavraia, which undergo polysporous sporogony in sporophorous vesicles, but differs from these genera in the mode of formation of sporoblasts and in the morphology of the sporophorous vesicles. Alignment and analyses of the small subunit ribosomal DNA sequences of T. hominis and several other polysporoblastic genera indicated that its closest phylogenetic relationships were with species of the genera Pleistophora and Vavraia, in line with morphological predictions. The type species of the latter two genera are Pleistophora typicalis and Vavraia culicis; these are parasites of fish and mosquitoes, respectively. These results suggest two possible routes and sources of infection to AIDS patients, these being perorally by ingestion of inadequately cooked fish or crustaceans or percutaneously during a bloodmeal taken by a haematophagous insect. Support for an insect source has been provided by recent detection of a microsporidium from mosquitoes in human corneal tissue.

Vairimorpha imperfecta n.sp., a microsporidian exhibiting an abortive octosporous sporogony in Plutella xylostella L. (Lepidoptera: Yponomeutidae).

The microsporidian genus Nosema is characterized by development in direct control with host cell cytoplasm, diplokaryotic nuclei throughout development and disporous sporogony. The genus Vairimorpha exhibits the same features plus an octoporous sporogony producing uninucleate spores in a sporophorous vesicle. A microsporidium from diamondback moth, Plutella xylostella, falls between Nosema and Vairimorpha in that it initiates but fails to complete the octosporous sequence in this host. The name Vairimorpha imperfecta n.sp. is proposed. Merogony is mainly by formation of buds from multinucleate meronts, the buds remaining attached in chains. Diplokaryotic spores measure 4.3 x 2.0 microns (fresh) and have 15.5 coils of the polar tube in 1 rank. The octosporous sporogony is aborted owing to irregular formation of nuclear spindles, incomplete cytoplasmic fission and bizarre deposition of electron-dense episporontal secretions. Phylogenetic analyses of the sequences of the small subunit rRNA genes of V. imperfecta and of several Nosema and Vairimorpha spp. place V. imperfecta in a clade with Nosema spp. from Lepidoptera rather than in the clade containing the more typical species of Vairimorpha. It is suggested that the ancestors of the Vairimorpha/Nosema complex of species exhibited both disporous and octosporous sporogonies, as does the type species of Vairimorpha, Vairimorpha necatrix. It would follow that true Nosema spp. have lost the ability to express an octosporous sequence and that V. imperfecta is in the process of losing it. It is proposed that the genera Nosema and Vairimorpha be placed in the same family Nosematidae Labbé 1899, rather than in separate families and orders as at present.

Nosema tyriae n.sp. and Nosema sp., microsporidian parasites of Cinnabar moth Tyria jacobaeae.

Nosema tyriae n.sp. was found in 63% of a population of Cinnabar moth larvae (Tyria jacobaeae). The infection was found in the gut wall, silk glands, and fat body and was probably generalized but appeared to be of low pathogenicity. Merogony and sporogony were by binary fission of diplokaryotic stages. Fresh spores were elongate, slightly pointed at the anterior end, and measured 4.7 x 2.0 microm. Ultrastructural features of special interest were 20-nm tubules connecting the surface of sporonts with host cell cytoplasm and, in the spores, a deeply domed polar sac, polaroplast consisting of closely packed longitudinally arranged membranes and loosely packed horizontally arranged membranes, and 10.5-14 coils of the polar tube in a single rank. The 16S rRNA genes of N. tyriae and Nosema bombycis from silkworms, Bombyx mori, differed by only six nucleotides and N. tyriae spores gave a moderately positive reaction with a monoclonal antibody raised to N. bombycis. N. tyriae was infective to B. mori but was less virulent than N. bombycis. However, no amplification product was obtained by PCR using N. tyriae DNA and primers considered to be specific for N. bombycis. Also, the spores of the two species are of entirely different shapes. A second diplokaryotic microsporidium, Nosema sp., found as a light infection in only one of the larvae had much smaller developmental stages and spores measuring 3.8 x 2.0 microm (fixed). Ultrastructurally it was distinguished by an abundance of dense membranes in cytoplasmic vesicles in both meronts and sporonts. Spores with up to 15 coils of the polar tube in irregular clusters or with about 12 coils in a single rank were observed in the tissues fixed from the one larva infected with this parasite. As this larva had been kept with N. tyriae-infected larvae for a few days before examination, it is possible that the two types of spores resulted from a double infection.