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J Clin Microbiol. Jan 2001; 39(1): 413–415.
PMCID: PMC87751

Causative Agent of Rhinosporidiosis

I have read the paper by Herr et al. (7) describing 18S ribosomal DNA (rDNA) in Rhinosporidium seeberi and its phylogenetic similarities with protoctistan fish parasites known as the DRIP clade. For extraction of DNA, the authors either dissected sporangia and purified them by centrifugation to remove human cells or used 100 mg of human tissue containing R. seeberi (7). This procedure without enzymatic digestion is insufficient for removing human cells that are always associated with sporangia (1, 3). Thus, contaminating human cells are the source of 18S rDNA amplified in this study (7).

We have isolated a prokaryotic cyanobacterium, a Microcystis sp. (a blue-green alga), from pond water samples where patients had been bathing. The same cyanobacterium, Microcystis, and its daughter cells termed nanocytes have been demonstrated in clinical samples (4). After gaining entry into the light-deprived environment in human epithelium, the photosynthetic cells of Microcystis differentiate into round bodies containing many Microcystis cells described by Herr et al. (7) as sporangia and spores.

We have successfully cultured the organism isolated from round bodies for the first time (2). Pure axenic cells were used for extraction of DNA. Our team has compared the DNA from Microcystis from pond water with the DNA from microbes found in clinical samples by using PCR, cloning, sequencing, and Southern hybridization. Our study demonstrates the presence of a prokaryotic cyanobacterium inside round bodies (unpublished results) but not 18S rDNA. Vanbreuseghm (12) had also suggested that R. seeberi produces precursors of chlorophyll and should be regarded as a pathogenic alga.

In the findings of Herr et al. (see Fig. 2A in reference 7), structures labeled as nuclei do not demonstrate a double membrane, while ribosome-like configurations are visible both outside and inside these “nuclei.” Actually, these are not nuclei but nanocytes of Microcystis encompassing naked prokaryotic DNA. The mitochondria illustrated in the work of Herr et al. (see Fig. 2A of reference 7) do not demonstrate two membranes. The mitochondria with flat cristae shown in Fig. 2B of this study (7) have two distinct membranes and are strikingly similar to those present in human cells. Since mitochondria magnified in Fig. 2B in reference 7 do not belong to Fig. 2A, their spatial relationship to sporangia or human cells and their source are not clear. It is noteworthy that mitochondria were observed only in intermediate sporangia (7), in which a large amount of host epithelial cells is present (see Fig. 5 in reference 9). Why were mitochondria, which are specialized for vital functions, not observed in young and mature sporangia? Why did previous investigators not find well-defined mitochondria? I am convinced that these mitochondria (7) are from human cells. On the basis of flat cristae in mitochondria in R. seeberi (see Fig. 2B in reference 7) and in Dermocystidium (10), Herr et al. (7) have suggested a relationship between R. seeberi and the DRIP clade. Flat cristae are not a distinctive feature of the DRIP clade but are common to most eukaryotes. The authors who studied the DRIP clade have themselves stated that mitochondria in Dermocystidium are like those in almost all animals and eumycota (10).

Herr et al. mention that I consider the causative organism an artifact of carbohydrate waste (7). I have never stated that R. seeberi is an artifact (1). The spheres of cellular waste (1) are now found to be polysaccharide reserves characteristic of cyanobacteria (5). Unexplained vacuoles, vesicles, and refractile bodies, as well as the concentric lamellated bodies in R. seeberi (8, 9, 11), correspond to cell inclusions (5) and photosynthetic membranes in cyanobacteria (6).

Finally, the conclusion of Herr et al. (7) that R. seeberi is related to the DRIP clade, based on 18S rDNA and mitochondria from human cells, is unwarranted. Supporting evidence drawn from superficial criteria such as spherical parasites, endospores, the inability to culture, and the aquatic habitat (7) has very little meaning. Inadequate knowledge about the various manifestations of the microbe, after acquisition of the pathogenic state, may also have led to the erroneous conclusions.

REFERENCES

1. Ahluwalia K B. New interpretations in rhinosporidiosis, enigmatic disease of the last nine decades. J Submicrosc Cytol Pathol. 1992;24:109–114. [PubMed]
2. Ahluwalia K B. Culture of the organism that causes rhinosporidiosis. J Laryngol Otol. 1999;113:523–528. [PubMed]
3. Ahluwalia K B, Bahadur S. Rhinosporidiosis associated with squamous cell carcinoma of the tongue. J Laryngol Otol. 1990;104:648–650. [PubMed]
4. Ahluwalia K B, Maheshwari N, Deka R C. Rhinosporidiosis: a study that resolves etiologic controversies. Am J Rhinol. 1997;11:479–483. [PubMed]
5. Allen M M. Cyanobacterial cell inclusions. Annu Rev Microbiol. 1984;38:1–25. [PubMed]
6. Golecki J R, Drews G. Supramolecular organization and composition of membranes. In: Carr N G, Whitton B A, editors. The biology of cyanobacteria. Oxford, England: Blackwell Scientific Publications; 1982. pp. 128–138.
7. Herr R A, Ajello L, Taylor J W, Arseculeratne S N, Mendoza L. Phylogenetic analysis of Rhinosporidium seeberi's 18S small subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade. J Clin Microbiol. 1999;37:2750–2754. [PMC free article] [PubMed]
8. Kannan-Kutty M, Teh E C. Rhinosporidium seeberi: an electron microscopic study of its life cycle. Pathology. 1974;6:63–70. [PubMed]
9. Kennedy F A, Buggage R R, Ajello L. Rhinosporidiosis: a description of an unprecedented outbreak in captive swans (Cygnus spp.) and a proposal for revision of the ontogenic nomenclature of Rhinosporidium seeberi. J Med Vet Mycol. 1995;33:157–165. [PubMed]
10. Ragan M A, Goggin C L, Cawthorn R J, Cerenius L, Jamieson A V C, Plourdes S M, Tand T G, Soderhall K, Gutell R R. A novel clade of protistan parasites near the animal fungal divergence. Proc Natl Acad Sci USA. 1996;93:11907–11912. [PMC free article] [PubMed]
11. Thianprasit M, Thagerngpol K. Rhinosporidiosis. In: McGinnis M R, Borgers M, editors. Current topics in medical mycology. Vol. 3. New York, N.Y: Springer Verlag; 1989. pp. 64–85. [PubMed]
12. Vanbreuseghm R. Ultrastructure of Rhinosporidium seeberi. Int J Dermatol. 1973;12:20–28. [PubMed]
Jan 2001; 39(1): 413–415.

AUTHORS' REPLY

Leonel Mendoza and Roger A. Herr
Medical Technology Program
Department of Microbiology
Michigan State University
322 N. Kedzie Lab.
East Lansing, Michigan 48824

We thank Dr. Ahluwalia for the opportunity to address her position on the prokaryotic nature of Rhinosporidium seeberi (1-4, 1-5). We differ with the arguments presented in her letter regarding our phylogenetic analysis of R. seeberi (1-7) and her assertion that this hydrophilic pathogen is a cyanobacterium and not a Mesomycetozoan. Our position is based on our studies (1-7) and a report by Fredericks et al. (1-6) which confirmed our phylogenetic analysis.

Dr. Ahluwalia's statement that the 18S small-subunit (SSU) rDNA that we isolated from R. seeberi's endospores and sporangia came from DNA of human origin is groundless and reflects little understanding of molecular microbiological procedures. An experienced molecular biologist would have readily noted that the human 18S SSU rDNA nucleotide sequences and our sequence (GenBank accession no. AF118851) are unrelated. In fact, as per our BLAST (Basic Local Alignment Search Tool) analysis, our sequence was not found in the human genome. Moreover, the sequence of R. seeberi published by Fredericks et al. (GenBank accession no. AF158369) (1-6), from a dog with rhinosporidiosis, is identical to our sequence. Both of these groups proved beyond doubt that their 18S SSU rDNAs came from the sporangia and endospores of R. seeberi. Fredericks et al. (1-6) did not contaminate their samples with canine DNA; neither could our sequence (1-7) possibly be of human origin. These two independent reports on the phylogenetic connection of R. seeberi with the Mesomycetozoans clearly indicate that Dr. Ahluwalia's prokaryotic theory is incorrect. There can be no reasonable doubt that R. seeberi is a eukaryotic organism not only on the basis of the two cited DNA studies (1-6, 1-7) but on the demonstration by numerous investigators of the presence of prominent nuclei and mitochondria in the sporangia of this pathogen (1-8, 1-9).

Dr. Ahluwalia's evolving concepts regarding the nature of R. seeberi have ranged widely during the past 8 years. In 1992, she concluded, on the basis of light microscopy, electron microscopy, and cytochemical studies of tissue from subjects with rhinosporidiosis, that “The round body is a unique structure composed of both plant and human material that is self-assembled in response to specific function pertaining to its elimination from the tissue” (1-1). In a follow-up paper (1-2), she stated that “This study provides unequivocal evidence against involvement of fungus in rhinosporidiosis.” “Two carbohydrates, namely defective proteoglycans synthesized intracellularly and an exogenous polysaccharide ingested through diet of tapioca constitute indigestible material in NB (nodular bodies) and scw (spheres of cellular waste).” In a 1994 publication, Ahluwalia et al. (1-3) concluded that “The so-called fungal sporangium was shown to be a unique body organized in host tissue for elimination of two indigestible carbohydrates, starch (possibly from tapioca) and defective proteoglycans (precursor of mucus).” The authors went on to say that “Dietary dry tapioca and chronic inflammation in undernourished individuals could lead to rhinosporidiosis.”

From this bizarre concept, Ahluwalia et al. in 1997 (1-4) went on to publish a new hypothesis on the etiologic agent of rhinosporidiosis. In this study, she announced that “We have been able to isolate the cyanobacterium Microcystis aeruginosa from water samples of ponds and rivers where patients of rhinosporidiosis were bathing. It is likely that this cyanobacterium is the causative agent of this disease.” Finally, in 1999 (1-5), she announced that “Observations based on laser-scanning confocal microscopy, light and electron microscopy confirm that a cyanobacterium Microcystis sp. is the causative agent of the disease. Rhinosporidiosis is the first human disease shown to be caused by a cyanobacterium.” Surprisingly, controls such as samples from normal people inhabiting the same areas where the water samples were collected and cultures of endospores and sporangia free of bacteria obtained after multiple washes and filtration steps were not included. It would also be interesting to probe M. aeruginosa with sera from patients with rhinosporidiosis, a key experiment that was also absent in Ahluwalia's studies. The mere isolation of the ubiquitous blue-green bacterium M. aeruginosa from pond and river waters in India does not have any significance with respect to the infections caused by the eukaryotic protist R. seeberi. Thus, the lack of controls in Dr. Ahluwalia's experiments is the source of her errors. This lapse undoubtedly led Dr. Ahluwalia to conclude that the mere isolation of M. aeruginosa from infected tissues was enough to prove her conjecture.

We disagree with Dr. Ahluwalia's statement that there are no other studies depicting mitochondria in R. seeberi. At least two independent studies showed these organelles within R. seeberi's sporangia (1-8, 1-9). In an effort to further clarify our finding, we have included Fig. 1-1. In this figure an intermediate sporangium (Fig. 1-1A, previously printed in reference 1-7) containing several nuclei, mitochondria, and a laminated body is shown. An enlargement of Fig. 1-1A presents details of the flat mitochondrial cristae of R. seeberi (Fig. 1-1B and C). The presence of a laminated body indicates that this is a healthy sporangium and that the mitochondria within the spherical body are not of human origin, as charged by Ahluwalia, but are attributable to R. seeberi.

FIG. 1-1
(A) Transmission electron microscopy photograph of a R. seeberi's intermediate sporangium, showing several mitochondria with flat cristae (black and white asterisks) and a laminated body (arrow). (B and C) An enlargement of the mitochondria in panel A. ...

Contrary to Dr. Ahluwalia's studies, our results have already been confirmed by other investigators (1-6). These researchers also concluded that R. seeberi is phylogenetically linked with the Mesomycetozoa (DRIP) clade. In contrast, we could not replicate her results when purified endospores and sporangia were cultured in a variety of media. We strongly recommend that Dr. Ahluwalia repeat our molecular studies with uncontaminated endospores and sporangia from patients with rhinosporidiosis in India to contest our data. However, the results achieved so far by two independent laboratories could well be the requiem to Dr. Ahluwalia's prokaryotic theory of R. seeberi.

REFERENCES

1-1. Ahluwalia K B. Plant molecular in human disease—a novel association. In: Wegmann R J, Wegmann M A, editors. Gene regulation and molecular aspects of muscle, liver, pancreas, connective tissue and plants. Vol. 5. Leuven, Belgium: Peeters Press; 1992. pp. 289–292.
1-2. Ahluwalia K B. New interpretations in rhinosporidiosis, enigmatic disease of the last nine decades. J Submicrosc Cytol Pathol. 1992;24:109–114. [PubMed]
1-3. Ahluwalia K B, Sharma N, Kacker S K, Deka R C. Association of tapioca and chronic inflammation with rhinosporidiosis. Indian J Otolayngol Head Neck Surg. 1994;3:25–27.
1-4. Ahluwalia K B, Maheshwari N, Deka R C. Rhinosporidiosis: a study that resolves etiologic controversies. Am J Rhinol. 1997;11:479–483. [PubMed]
1-5. Ahluwalia K B. Culture of the organism that causes rhinosporidiosis. J Laryngol Otol. 1999;113:523–528. [PubMed]
1-6. Fredericks D N, Jolley J A, Lepp P W, Kosek J C, Relman D A. Rhinosporidium seeberi: a human pathogen from a novel group of aquatic protistan parasites. Emerg Infect Dis. 2000;6:273–282. [PMC free article] [PubMed]
1-7. Herr R A, Ajello L, Taylor J W, Arseculeratne S N, Mendoza L. Phylogenetic analysis of Rhinosporidium seeberi's 18S small subunit ribosomal DNA groups this pathogen among members of the protoctistan Mesomycetozoa clade. J Clin Microbiol. 1999;37:2750–2754. [PMC free article] [PubMed]
1-8. Teh E C, Kannan-Kutty M. Rhinosporidium seeberi: spherules and their significance. Pathology. 1975;7:133–137. [PubMed]
1-9. Thianprisit M, Thagerngpol K. Rhinosporidiosis. Curr Top Med Mycol. 1989;3:61–85.

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