Purification of the Endospores and Sporangia of Rhinosporidium Seeberi on Percoll Columns

Human rhinosporidial tissue was used as the source of the various developmental stages of Rhinosporidium seeberi - endospores with electron dense bodies, juvenile, and immature sporangia. After homogenisation in phosphate buffered saline (PBS) and removal of tissue fragments by centrifugation, the rhinosporidial bodies were isolated on centrifuged Percoll columns with gradients of densities or on triple-layered columns of varying density. The separated bands, after repeated washing in PBS gave bodies free from human tissue as shown on Leishman and PAS staining and indirect immunofluorescence with rabbit and human patients' anti-rhinosporidial sera. Sonicates of these bodies were tested on agarose gel for precipitation with antisera, and on SDS-PAG electrophoresis and Coomassie Blue staining. Percoll columns were shown to be capable of isolating these stages of R. seeberi, free from human tissue and contaminating bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anti-Rhinosporidial Antibody Levels in Patients with Rhinosporidiosis and in Asymptomatic Persons,in Sri Lanka

The only report hitherto, from India in 1982, on anti-rhinosporidial antibody levels in patients with rhinosporidiosis recorded that antibody was not detected in Indian patients. The present report describes the use of the dot-ELISA assay of serum anti-rhinosporidial IgG, IgM and IgA and salivary sIgA in patients with diverse clinical presentations, in rural asymptomatic persons who had bathed in ground waters that probably harboured the causative pathogen, Rhinosporidium seeberi, and in laboratory persons who were exposed to R. seeberi. Ultrasonic extracts of purified endospores and sporangia of R. seeberi were used as antigen. The geometric mean (reciprocal) titres of serum antibody detected in patients were IgM 142.1, IgG 178.5, IgA 84.6, with ranges of 0-640, 30-960 and 0-160 respectively, salivary sIgA titres ranged from 0 to 18 with a mean of 4.6. The levels of antibody had no correlation with the site, the number of sporangia, duration and recurrence of the disease. Asymptomatic persons from the same endemic area as patients showed mean titres of IgM 89.6, IgG 69.1, IgA 95.5, with salivary sIgA titres of 3.1. Asymptomatic personnel who had been working in a laboratory where rhiniosporidial work was being done, showed mean titres of 169.6 IgM, 62.8 IgG, and 6.5 salivary sIgA. These results indicate that an anti-rhinosporidial antibody response occurs in rhinosporidial patients, as well as in asymptomatic persons who were exposed to R. seeberi in the environment. Anti-R. seeberi antibody does not appear to be protective in rhinosporidiosis since appreciable titres were present in patients with recurrent, single, multiple or disseminated lesions of long duration.     

Cell-mediated immune responses (CMIR) in human rhinosporidiosis

Cell mediated immune responses (CMIR) to Rhinosporidium seeberi in human patients with rhinosporidiosis have been studied. With immuno-histochemistry, the cell infiltration patterns in rhinosporidial tissues from 7 patients were similar. The mixed cell infiltrate consisted of many plasma cells, fewer CD68+ macrophages,a population of CD3+ T lymphocytes, and CD56/57+ NK lymphocytes which were positive for CD3 as well. CD4+ T helper cells were scarce. CD8+suppressor/cytotoxic-cytolytic cells were numerous. Most of the CD8+ cells were TIA-l+ and therefore of the cytotoxic subtype. CD8+ T cells were not sub-typed according to their cytokine profile; 1L2, IFN-γ (Tcl); IL4, ILS (Tc2).In lympho-proliferative response (LPR) assays in vitro, lymphocytes from rhinosporidial patients showed stimulatory responses to Con A but lymphocytes from some patients showed significantly diminished responses to rhinosporidial extracts as compared with unstimulated cells or cells stimulated by Con A, indicating suppressor immune responses in rhinosporidiosis. The overall stimulatory responses with Con A suggested that the rhinosporidial lymphocytes were not non-specifically anergic although comparisons of depressed LPR of rhinosporidial lymphocytes from individual patients, to rhinosporidial antigen with those to Con A, did not reveal a clear indication as to whether the depression was antigen specific or non-specific. The intensity of depression of the LPR in rhinosporidial patients bore no relation to the site, duration, or the number of lesions or whether the disease was localized or disseminated. Rhinosporidial extracts showed stimulatory activity on normal control lymphocytes, perhaps indicating mitogenic activity. These results indicate that CMIR develops in human rhinosporidiosis, while suppressed responses are also induced. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cell-mediated immune responses (CMIR) to Rhinosporidium seeberi in mice

There is no published data on Cell Mediated Immune Responses in experimental animals to Rhinosporidium seeberi the causative agent of human and animal rhinosporidiosis. The quantitative mouse foot-pad model was used to assay the Delayed-Type Hypersensitivity (DTH) cell-mediated immune response to extracts of purified endospores and sporangia of R. seeberi. Histological examination was used to confirm that the foot-pad reactions were compatible with DTH reactions in the mouse. We report that sonically disintegrated rhinosporidial endospores/sporangia induced DTH responses in the foot-pads of sensitized mice which were comparable in intensity and histological profile to that induced by sheep red blood cells in SRBC sensitized mice. Anti-rhinosporidial antibody was also induced. Filtrates of the soluble antigens in sonicated suspensions failed to evoke a DTH-foot-pad (DTH-FP) response in sensitized mice although an anti-rhinosporidial antibody response to this preparation was detected. Prolonged pre-treatment with sonicated suspensions of endospores and sporangia resulted in a decrease of DTH reactivity as compared with reactions following pre-treatment of a shorter duration. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

A pilot plant for detoxification of aflatoxin B1-contaminated coconut oil by solar irradiation

Summary In coconut oil naturally contaminated with aflatoxin B1, more than 85% of the toxin is present in the soluble form, the remainder occuring in the sediment. This aflatoxin is detoxified when the oil, in a static layer less than 15 mm thick is exposed to solar radiation. A pilot plant, designed to take account of the viscosity and flow characteristics of the oil, was constructed for the exposure of thin layers of oil (2 mm or less) flowing under gravity. At aflatoxin concentrations between 166 and 1250 jug/kg, 75% of the toxin was degraded on exposure to solar radiation of 10 cal/cm²; total detoxification was achieved on repeated exposure. The naturally contaminated coconut oil after exposure to solar radiation did not contain any residual aflatoxins or fluorescent compounds which might have been derived from original aflatoxin B1.

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Resumen Planta pilota para el tratramiento de aceite de coco contaminado con aflatoxina B1 mediante radiation solar y centrifugado En aceite de coco contaminado de forma natural con aflatoxina B1, más del 85% de la toxina se encuentra en forma soluble, el resto permaneciendo en el sedimento. Esta aflatoxina pierde sus propiedades tóxicas cuando se expone el aceite en forma de capa estática de 15 mm de grosor a la radiación solar. Se construyó una planta piloto diseñada teniendo en cuenta la viscosidad y las caracteristicas de fluidez del aceite de forma que pudieran exponerse a la radiación solar capas de aceite muy finas (2 mm o menos) fluyendo gracias a la gravedad. Cuando aceite conteniendo aflatoxina en proporciones entre 166 y 1250 /kg se expuso a una radiación solar de 10 cal./cm² la toxina se degradó en un 75%. La detoxificación total se obtuvo mediante repetición del proceso. El aceite de coco contaminado de forma natural, una vez expuesto a la radiación solar no contenía aflatoxinas residuales ni compuestos fluorescentes potencialmente derivados de la aflatoxina B1 contenida originalmente.

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Résumé Usine-pilote pour la détoxification, par irradiation solaire et centrifugation, de l'huile de coprah contaminée par l'aflatoxine B1 Dans l'huile de coprah spontanément contaminée par l'aflatoxine B1, plus de 85% de la toxine est présente sous forme soluble, le reste se trouvant dans le sédiment. Cette aflatoxine est détoxifiée lorsque l'huile est exposée à la radiation solaire en couche statique de moins de 15 mm d'épaisseur. Une usine-pilote, conçue en tenant compte de la viscosité et de l'écoulement de l'huile, a été construite pour irradier une mince couche d'huile (2 mm ou moins) s'écoulant par gravité. Pour des concentrations en aflatoxine allant de 166 à 1250 g/kg, 75% de la toxine est dégradée par exposition à une irradiation solaire de 10 cal./cm² et, en répétant le traitement, on obtient une détoxification complète. Après exposition à la radiation solaire, l'huile de coprah contaminée spontanément ne contient plus d'aflatoxine résiduelle, ni de composés fluorescents dérivés de l'aflatoxine B1 originelle.

     

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.     

Non-toxicity to ducklings of solar-irradiated coconut oil contaminated with aflatoxin

Summary Pure aflatoxin Bl caused reduction in rate of weight gain, more than 50% mortality, bile duct hyperplasia and focal necrosis when fed to one-day-old veluvi ducklings for seven days. The same amount of aflatoxin B1 in coconut oil was solar irradiated in a pilot plant. The oil was then extracted for aflatoxins and the extracts fed to ducklings so that they would receive the same dose as birds fed with pure aflatoxin Bl. The pattern of weight gain in the birds fed with extracts of toxic oil after irradiation was the same as those of birds in control groups fed with non-toxic oil after irradiation or the vehicle, propylene glycol, alone. The birds showed no mortality or histopathological changes attributable to aflatoxins. Solar irradiation appeared to remove the toxicity of aflatoxin B1 in coconut oil, confirming TLC analysis of the irradiated oil.

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Aceite de coco contaminado con aflatoxinas, sometido a radiación solar, sin toxicidad para crias de pato

Resumen Se alimentaron crias de pato de un dia de edad de la raza veluvi durante siete dias con aflatoxina B1 pura. Como consecuencia se observó una reducción en la tasa de incremento de peso, más de un 50% de mortalidad e hyperplasia de los conductos biliares con necrósis focal. La misma cantidad de aflatoxina B1 en aceite de coco se sometió a radiación solar en una planta piloto. A continuación se extrajo el aceite para la obtención de aflatoxinas. Los extractos se dieron a crías de pato de forma que recibieran la misma dósis que las que se alimentaron con aflatoxina B1 pura. El patrón seguido por el incremento de peso de las crías alimentadas con los extractos del aceite tóxico irradiado fue el mismo que el obtenido por las crias alimentadas con aceite no tóxico irradiado y por otras que lo fueron con tan sólo el vehículo: propilenglicol. No se observaron variaciones en la mortalidad ni en la histopatología de las crías que fueran atribuíbles a aflatoxinas. La radiación solar parece eliminar la toxicidad de la aflatoxina B1 en aceite de coco, confirmando el análisis TLC (cromatografía de capa fina) del aceite irradiado.

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Non-toxicité pour les canetons de l'huile de coco contaminée par une aflatoxine et traitée par irradiation solaire

Résumé Administrée oralement à des canetons veluvi pendant sept jours, l'aflatoxine B1 pure détermine une réduction du gain de poids, une hyperplasie des canaux biliaires et des nécroses focales. La même quantité d'aflatoxine B1 dans de l'huile de coco a été irradiée par le soleil dans une usine pilote. Les aflatoxines ont été extraites et les extraits administrés à des canetons de façon à ce que ceux-ci reçoivent la même dose que des oiseaux traités par l'aflatoxine Bl pure. Les gains de poids des oiseaux traités par les extraits de l'huile toxique irradiée sont identiques à ceux des oiseaux témoins des groupes de contrôle ayant reçu de l'huile non-toxique irradiée, ou le véhicule seul (propylène glycol). Les oiseaux n'ont présenté aucune mortalité nimanifestation histopathologique attribuable aux aflatoxines. L'irradiation solaire parait éliminer la toxicité de l'aflatoxine B1 dans l'huile de coco, ce qui confirme l'analyse par TLC de l'huile irradiée.

     

In vitro studies on the mechanisms of endospore release by Rhinosporidium seeberi

Studies of Rhinosporidium seeberi have demonstrated that this organism has a complex life cycle in infected tissues. Its in vivo life cycle is initiated with the release of endospores into a host's tissues from its spherical sporangia. However, little is known about the mechanisms of sporangium formation and endospore release since this pathogen is intractable to culture. We have studied the in vitro mechanisms of endospore release from viable R. seeberi's sporangia. It was found that watery substances visibly stimulates the mature sporangia of R. seeberi to the point of endospore discharge. The internal rearrangement of the endospores within the mature sporangia, the opening of an apical pore in R. seeberi's cell wall, and the active release of the endospores were the main features of this process. Only one pore per sporangium was observed. The finding of early stages of pore development in juvenile and intermediate sporangia suggested that its formation is genetically programed and that it is not a random process. The stimulation of R. seeberi's sporangia by water supports the epidemiological studies that had linked this pathogen with wet environments. It also explains, in part, its affinities for mucous membranes in infected hosts. The microscopic features of endospore discharge suggest a connection with organisms classified in the Kingdom Protoctista. This study strongly supports a recent finding that placed R. seeberi with organisms in the protoctistan Mesomycetozoa clade. This revised version was published online in June 2006 with corrections to the Cover Date.