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Microsporidia are intracellular parasites that are common in invertebrates. Taxonomic classification is mostly restricted to morphologic and physiologic data. Limited data are available about taxonomic classification using DNA-sequence data for analysis. We examined the small-subunit (SSU) rDNA, the intergenic spacer (ITS) region, and a part of the large-subunit (LSU) rDNA of Nosema algerae, a parasite of mosquitoes, taken from a laboratory colony of Anopheles stephensi. Target gene amplifications were done by polymerase chain reaction (PCR) and, after cloning, DNA fragments were sequenced. The SSU-rDNA sequence obtained was aligned with several other microsporidian SSU-rDNA sequences available from the GenBank or EMBL data bases and was analyzed by different methods. On the basis of the results of our phylogenetic analysis, we suggest that our N. algerae isolate is not closely related to other microsporidia belonging to the genus Nosema.

Microsporidia form a large and ubiquitous group of obligately intracellular parasitic eukaryotes, increasingly recognized as pathogens in humans. Transmission of invertebrate microsporidia to mammals has been considered impossible because temperature seemed to be a limiting factor for development. Nosema algerae, a microsporidian of anopheline mosquitoes, was cultured in human muscle fibroblasts at temperatures of 31 degrees C and 38 degrees C. This is the first record of an invertebrate microsporidian developing in human cells at a temperature above 36 degrees C. The ultrastructure of N. algerae growing in human muscle fibroblasts is similar to that of Brachiola vesicularum, a microsporidian species previously described in the muscle of an AIDS patient.

By using different staining techniques, 479 stool specimens from 212 diarrheic patients with AIDS were examined for microsporidian spores. Calcofluor fluorescence staining of 119 specimens revealed fluorescent ovoid structures of microsporidian size. Staining of these samples according to the method of Weber et al. (R. Weber, R. T. Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin, and G. S. Visvesvara, N. Engl. J. Med. 326:161-166, 1992) with trichrome produced six specimens with pinkish spores containing the characteristic microsporidian belt-like structure. The 6 specimens were processed for transmission electron microscopy, as were another 21 specimens which did not present the belt-like structure after trichrome staining but which looked highly suspicious after fluorescence staining. In these 21 samples, only fungal spores and, particularly, bacterial Clostridium spores were demonstrated, whereas in the 6 samples diagnosed positive after trichrome staining, the existence of microsporidia could be verified by electron microscopy. Based on our observations, we propose that the belt-like structure seen with the Weber stains in microsporidian spores corresponds to structures existing in priming-stage spores. The results suggest that routine microscopical fecal diagnosis for microsporidian infection should include a screening by fluorescence staining and, subsequently, a confirmatory viewing of fluorescence-positive samples after trichrome staining.

As a member of the phylum Microspora, Nosema algerae is a small obligate intracellular parasite. Its free invasive stage is a spore with a characteristic cellular organization, including an apically anchored polar tube that serves as a tool for the transmission of genetic material into the host cell. By detailed electron micrographic documentation of the spore ultrastructure we present the aspects related to the biologic process of spore extrusion. Our ultrastructure findings confirm that the extrusion process of microsporidian spores is based on extreme changes in their organization. This study is the first complete ultrastructural documentation of N. algerae concerning the extrusion process, which can be subdivided into different stages: the breakdown of the microsporidian cellular compartmentation; the filling of a preformed polar tube with modified sporoplasm; the uncoiling of the polar tube, which in this stage has reached its final length; and, finally, its extrusion and screw-like movement.

Laser treated rat fundi were observed angiographically and with the fluorescence microscope for 3 months. Angiographically diffusions have been observable up to about one week post laser, fluorescence microscopically, however, for the whole observation time of 3 months. From the 2nd week, these diffusions were produced mainly by neovascularizations which angiographically were not visible. They started from the choroid and penetrated into the outer part of the retina.

For contributing to the question of an existence of a diffusion of the unbound part of the fluorescein between choroid and retina in fluorescein angiography, on pigmented rats, after intravenous injection of Fluorescein sodium and Rhodamine B respectively, comparing observations were effectuated. Rhodamine B in its unbound form showed a distinct diffusion between choroid and retina and coloured all retinal layers in the late phases whereas in the case of the Fluorescein sodium, only a poor diffusion was observable. The similar spectral bands of the primary and secondary fluorescences render difficult the observation of the behaviour of the fluorescein dye.

In addition to previous fluorescence-microscopic investigations on ocular barriers, quantitative fluorescence-photometric measurements on the eye tissues of albinotic rats were performed. The eyes were enucleated 5-30 min after intravenous injection, and tissue samples were compared with analogous eye tissues of untreated animals. The results were (I) different increases of measured data in the region of endothelium and stroma of the cornea, of vessels and stroma of the ciliary body and iris, of choroid and choriocapillaris, as well as of pigment epithelium and retina vessels; (II) no increase of the photometer values was observed in the epithelium of the cornea and in all other external and internal layers of the retina. It is to be assumed that in contrast to the tissues and vessels mentioned in (I), no measurable amounts of fluorescein have penetrated into the retina layers mentioned in (II). Although the fluorescence-photometric method may not be absolutely conclusive, it could be established that measurements are more exact than the qualitative method performed by fluorescence-microscopic observation.

We conceived and manufactured a holder for rats. It is a useful little apparatus for some experiments on rats as well as for its fundus photography, angiographies included. The utility of our device is discussed and illustrated.

The effects of laser coagulation on rat fundi were studied angiographically and with the fluorescence microscope. The coagulations were performed using constantly 300 mW and 0.2 second exposure time and 50 microns spot diameter. We observed the animals for 3 months, after which we could still observe dye diffusion by fluorescence angiography. With the fluorescence microscope we could localize these diffusions as originating from neovascularisation starting from the choroid. These new vessels penetrated subsequently into the exterior retinal layers. At the beginning of our observation time, the retinal vessels showed some sinuosities, dilatations and solitary stenosis, but no disturbance of circulation was demonstrable.

The corneal neovascularization produced by NaOH burns was examined in two groups of pigmented rabbits following laser treatment. The treatment was carried out with energy level 500 mW, spot diameter 200 micron, and exposure time 1 s throughout. In the first group, a single newly formed vessel was coagulated in each case. Subsequent fluorescein angiography invariably showed an incomplete occlusion of the vessel. In the second group, we coagulated a section of the neovascularization network at its origin in the corneal limbus. After 48 h, fluorescein perfusion was once again observed, but the vessels were predominantly finer than before. Fluorescence microscopy revealed a rich neovascularization of the more superficial layers of the corneal stroma. To be successful, laser treatment must involve both supplying and draining vessels.

3 weeks after NaOH cauterization of rat corneas, the newly formed vessels were angiographically investigated by means of FITC dextran fractions. An angiographic picture similar to that of fluorescein Na was observed after intravenous administration of FD 3. A minimum diffusion of FD 40 could only be detected in the area of the distal terminal loops of the newly formed corneal vessels. FD 70 and FS 150 shoed an angiographically similar behaviour with no diffusion in the whole area of neovascularization.

In pigmented rabbits we investigated the much-used drugs pilocarpine, atropine, and tropicamide (Mydriaticum, Roche) with regard to their possible influence on the permeability of newly formed corneal vessels. Sodium fluorescein was chosen as test substance. Corneal vessels were produced by introcorneal injection of 0.1 N NaOH. Their development required 2--3 weeks and their growth about 4--6 weeks. A 2-week period of stability followed and was used for the local application of the above drugs. An evident influence on the diffusion of fluorescein could not be ascertained. Tropicamide induced a narrowing of the dye column in the corneal vessels. This phenomenon may be due to a narrowing of supplying vessels. In all cases, dye diffusion took place especially at the peripheral loops of the corneal vessels.

After local application of gentamicin, dexamethasone and tropicamide to rat eyes, no effect on the permeability or the caliber of retinal vessels was observed, nor any diffusion of fluorescein from the choriocapillaris to the outer retinal layers. The qualitative assessment of fluorescence microscopic preparations (and photographic reproductions of them) is relatively unreliable and has led to confusion in the literature. The results presented here, which to some extent contradict earlier findings published in 1971, are discussed. The authors expect to obtain more accurate results from the quantitative method which they are now using.