Rhinosporidium seeberi Nuclear Cycle Activities Using Confocal Microscopy.

Rhinosporidium seeberi is an uncultivated Ichthyosporean infecting animals, including humans. Recent studies suggested R. seeberi undergoes synchronized nuclear division without cytokinesis. We used confocal microscopy to investigate R. seeberi nuclear division cycles in formalin-fixed tissues stained with DAPI and phalloidin. We report that R. seeberi nuclei in juvenile and intermediary sporangia synchronously divided without cytokinesis. Intermediary sporangia display numerous 3-4 µm nuclei at different mitotic stages as well as a thick inner layer with strong affinity for phalloidin. Mature sporangia showed numerous 5-12 µm cell-walled endospores, each containing a 2-4 µm in diameter nucleus. Phalloidin did not bind to the inner layers of mature sporangia or endospores. The development of a "germinative zone" in the inner layer of mature sporangia containing hundreds of nuclei was also confirmed. This study establishes that during the R. seeberi life cycle synchronous nuclear divisions without cytokinesis takes place, resulting in the formation of thousands of nuclei. Cytokinesis, on the other hand, is a 1-time event and occurs in the latest stages of intermediate sporangia, after the formation of thousands of nuclei and just before mature sporangia development.

Nasal rhinosporidiosis.

Rhinosporidiosis is a rare chronic granulomatous infection caused by Rhinosporidium seeberi. It affects mainly the mucosa of the nose, nasopharynx, palate, conjunctiva and the urethra. A seven-year-old girl presented with intranasal polypoid growth with recurrent nose bleeding for one year. Excision biopsy was done, and the tissue was subjected to routine histological processing and stained with hematoxylin and eosin stains with additional mucicarmine special stain. Variable-sized sporangia containing magenta-colored spores and capsule were observed. We hereby present a rare infective disease diagnosed nine years after the first reported case in our center.

Rhinosporidiosis in a domestic cat.

Rhinosporidiosis was diagnosed in a domestic shorthair cat from a suburb of Washington DC, USA. The clinical presentation of protracted sneezing and epistaxis was associated with a polypoid lesion in the right nostril. Light microscopic examination revealed a polypoid lesion with numerous sporangia containing maturing endospores. Free endospores were present in the stroma of the polyp and lumen of the nasal cavity. Transmission electron microscopy revealed ultrastructural features typical of Rhinosporidium seeberi. The case was followed clinically for a total of 70 months and there were five attempts at surgical excision. This is the first reported case of rhinosporidiosis in a domestic cat.

Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites.

Rhinosporidium seeberi, a microorganism that can infect the mucosal surfaces of humans and animals, has been classified as a fungus on the basis of morphologic and histochemical characteristics. Using consensus polymerase chain reaction (PCR), we amplified a portion of the R. seeberi 18S rRNA gene directly from infected tissue. Analysis of the aligned sequence and inference of phylogenetic relationships showed that R. seeberi is a protist from a novel clade of parasites that infect fish and amphibians. Fluorescence in situ hybridization and R. seeberi- specific PCR showed that this unique 18S rRNA sequence is also present in other tissues infected with R. seeberi. Our data support the R. seeberi phylogeny recently suggested by another group. R. seeberi is not a classic fungus, but rather the first known human pathogen from the DRIPs clade, a novel clade of aquatic protistan parasites (Ichthyosporea).

Phylogenetic analysis of Rhinosporidium seeberi's 18S small-subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade.

For the past 100 years the phylogenetic affinities of Rhinosporidium seeberi have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from R. seeberi. Using phylogenetic analysis, by parsimony and distance methods, of R. seeberi's 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium), near the animal-fungal divergence. Our phylogenetic analyses also indicate that R. seeberi is the sister taxon of the two Dermocystidium species used in this study. This molecular affinity is remarkable since members of the genus Dermocystidium form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of R. seeberi to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group's phylogenetic association within the Eucarya.

Immunolocalization of an endogenous antigenic material of Rhinosporidium seeberi expressed only during mature sporangial development.

We investigated the immunolocalization of Rhinosporidium seeberi's antigens using sera from individuals infected with R. seeberi and tissue from Sri Lankan patients with rhinosporidiosis. The tissues were fixed in LR white resin, thin sectioned fixed onto nickel grids and evaluated by transmission electron microscopy for the presence of R. seeberi's sporangia. The tissue samples were reacted with the patients's sera and then labeled with protein A colloidal gold (PACG) for immunolocalization. It was found that the PACG had fixed to antibodies that specifically recognized an internal electron lucent layer situated immediately under the mature sporangium's wall. Strikingly, the endospores, the juvenile and intermediate sporangia did not undergo PACG labeling. This study found that the expression of this antigen occurs only in the final developmental stages of R. seeberi's mature sporangia. Our data may explain why circulating antibodies to R. Seeberi were not detected before in studies that used endospores as antigen in immunoassays. This is the first report in which an antigenic material with a potential role in the immunology of rhinosporidiosis has been detected.

Rhinosporidiosis: a description of an unprecedented outbreak in captive swans (Cygnus spp.) and a proposal for revision of the ontogenic nomenclature of Rhinosporidium seeberi.

Rhinosporidiosis is a mucocutaneous zooanthroponotic disease caused by Rhinosporidium seeberi, a fungal-like organism of uncertain classification with an unknown mode of transmission. Over a 3 year period, 41 captive swans (Cygnus olor and C. atratus) developed conjunctival and cutaneous polypoid lesions diagnosed as rhinosporidiosis by histopathological examination including light and electron microscopy. Investigation of this avian outbreak, the first of its kind, provides additional insight into the epidemiology of this enigmatic aetiologic agent, which has yet to be isolated and cultivated in vitro. The occurrence of rhinosporidiosis in swans supports an aquatic environment as the reservoir for R. seeberi, which is often associated with exposure to water. We report the first known occurrence of rhinosporidiosis in 41 captive mute (C. olor) and Australian black (C. atratus) swans dwelling on a lake in a Central Florida city. Additionally, we review the development stages of R. seeberi and propose a revision in its ontogenic nomenclature to reflect its probable taxonomic classification as a member of the kingdom Fungi.

New interpretations in rhinosporidiosis, enigmatic disease of the last nine decades.

Fungal etiology is widely quoted for the disease rhinosporidiosis. Identity of the fungal sporangium and its relationship with the disease have baffled medical scientists and mycologists for several decades. This study provides unequivocal evidence against involvement of fungus in rhinosporidiosis. The so-called sporangium is found to be a unique body containing residue-loaded lysosomal bodies ('spores') for elimination from the system. 'Sporangia' have been redesignated nodular bodies (NB) and 'spores' as spheres of cellular waste (scw). Two carbohydrates, namely defective proteoglycans synthesized intracellularly and an exogenous polysaccharide ingested through diet of tapioca constitute indigestible material in NB and scw. Polysaccharide in NB which has beta, 1-4 glycosidic bonds between mannose residues is not degraded by gastrointestinal enzymes nor in intracellular lysosomes which break only alpha-glycosidic bonds. A link between NB and dry tapioca has been deduced. Rhinosporidiosis is a complex phenotype with perhaps no parallel in medical science. This report erases 99 years (1892-1991) of controversies regarding 'causal organism' of rhinosporidiosis.

Rhinosporidium seeberi: light, phase contrast, fluorescent and scanning electron microscopic study.

Phase contrast microscopic study indicated the multilayered structure of the sporangial wall of R. seeberi while the scanning electronmicroscopic study revealed a trilaminated wall compared to a thick double walled light microscopic structure. The scanning electronmicroscopy revealed the spores of varying sizes which were found either discretely or in groups interconnected and seen attached to the inner aspect of the sporangial wall. Autofluorescence of sporangia and spores was observed under microscope. Acridine orange staining revealed the presence of DNA materials in the spore and sporangia.

Rhinosporidiosis associated with squamous cell carcinoma in the tongue.

The peritumoural region of a squamous cell carcinoma of the tongue when examined with light and electron microscope showed nodular bodies in the submucosa with all the distinctive features of 'sporangium and 'spores' of rhinosporidiosis. The occurrence of rhinosporidiosis in the tongue along with malignancy has not been reported hitherto. Some interesting observations and causal relationships are discussed.

Culture of Rhinosporidium seeberi: preliminary report.

Every year 400 to 450 cases of Rhinosporidium are reported from Trivandrum Medical College. Twenty-five swabs were collected from patients suffering from Rhinosporidiosis and cultured in standard media. Positive results were obtained in 23 cases. The conidia produced from the colony were compared with the structures obtained from the patient material. Light microscopy using histopathological techniques were used. The consistent appearance of the organism in patient material, the repeatability of growth in subcultures and the negative growth in controls indicated that the organism grown in culture is the causative agent of the disease. The effect of parameters like pH, temperatures, etc, were also studied.

Rhinosporidiosis.

Rhinosporidium seeberi, the causative organism of rhinosporidiosis of the nasal mucosa and skin was reviewed with regard to its pathogenesis and histopathology, histochemistry, ultrastructure, life cycle, and cultivation. The pathological findings from infected tissues reveal a granulomatous reaction comprising mixed cell granuloma, pseudocystic abscesses, fibrosis around the causative organism (R. seeberi), and transepidermal elimination. The cell walls of trophocytes and sporangia exhibit the presence of cellulose. The spore wall is encapsulated with granular fibrillary substances consisting of acid mucopolysaccharides. Spheroid bodies have proved to be DNA surrounded by a thin membrane-bound layer. In the cytoplasm of the organism, various substances can be detected by histochemical methods (e.g., glycogen, glycoprotein, acid mucopolysaccharides, neutral lipids, and phospholipids). The walls of the sporangia are found to be trilaminated, whereas those of trophocytes are bilaminated. There is a myriad of curvilinear structures around the outer wall of both forms. The ultrastructure of a trophocyte shows it to be comprised of sporoblasts containing oval or round membrane-bound nuclei with nucleoli, mitochondria, endoplasmic reticulum, chromatin granules, vacuoles, lipid bodies, and spherules. We suggest that the multilamellar bodies are precursors of trophocytes and sporangia. Abortive trophocytes without cytoplasmic organelles are seen, and they collapse at the end of the maturation process. Rhinosporidium seeberi fails to grow in any of the artificial media used but can be maintained through its life cycle in tissue cultures.

Rhinosporidiosis at King Edward VIII Hospital, Durban--1976-1985. A report of 6 cases.

Six cases of the fungal infection, rhinosporidiosis, are documented. Clinical, light microscopic and electron microscopic features of the causative organism (Rhinosporidium seeberi) are presented.

Rhinosporidiosis: ultrastructural study of an infection in South Carolina.

We have morphologically described and ultrastructurally analyzed Rhinosporidium seeberi, the causative agent of rhinosporidiosis, obtained from a nasal polyp of a man who had never traveled to India or Ceylon. The morphology, endosporulation phases, and cell wall were similar to those in previously described infections. A common etiology is suggested and potential therapy is discussed.

Conjunctival Rhinosporidiosis. Light and electron microscopic study.

Two cases of conjunctival rhinosporidiosis were studied by light and electron microscopy. Two distinct phases of the tissue life cycle were present: trophic and endosporulating. Young trophocytes contained a single nucleus. As the trophocyte matured chromatin was dispersed throughout the cyst. During the next phase of the life cycle, sporangial cyst walls acquired a new inner layer that appeared to give rise to endospores. Histochemical and ultrastructural features of Rhinosporidium seeberi are consistent with it being a fungus. Complete surgical excision of the lesion is the only known method to eradicate the infection.

Rhinosporidiosis in The Netherlands. A case report including ultramicroscopic features.

A case of rhinosporidiosis in a 34-year-old male is presented. The histological findings are discussed.