Patricia Bonilla

Removal of pathogens from wastewaters by the root zone method (RZM)

Full-scale and laboratory-scale artificially constructed reed beds utilising the root zone method (RZM) of wastewater treatment were investigated for their removal efficiencies of a range of pathogenic microorganisms. Performances were compared for RZMs employing different hydrophytes, planting substrates and climatic conditions. All pathogens were removed by the RZM to some extent. Planted beds generally performed better than unplanted beds although the type of hydrophyte used was not significant. Gravel beds were more efficient than soil beds in the removal of protozoan pathogens and of helminth eggs; numbers of these pathogens were below levels of detection in gravel bed effluents. For faecal coliforms the type of planting substrate was not significant, providing hydrophytes were present. Climatic conditions made little difference to treatment efficiency although seasonal variation was observed in temperature climates, where the removal of faecal coliforms declined in the winter months, and in subtropical climates where higher numbers of free-living amoebae were recorded during the summer. It was concluded that the RZM is a suitable method for the treatment of domestic wastewaters in tropical and subtropical regions of Mexico.

Amoebae isolated from the atmosphere of mexico city and environs

A protozoological analysis was performed from June to August, 1982 to isolate small free-living amoebae from the atmosphere by using an air vacuum sampler and several filters. Monoxenic medium (nonnutritive agar plus Escherichia coli) and axenic medium (De Jonckheere, 1977) were used to culture the isolates. The species isolated included Naegleria sp. Alexeieff emend. Calkins, Acanthamoeba polyphaga Puschkarew, Vahlkampfia jugosa Page, Acanthamoeba astronyxis Ray and Hayes, Acanthamoeba castellanii Douglas, Acanthamoeba culbertsoni Singh y Das, Vahlkampfia ustiana Page, Saccamoeba stagnicola Page, Hyalodiscus sp. Hertwig and Lesser, Platyamoeba placida Page, Rugipes sp. Schaeffer, Vannella platypodia Gläser, Vahlkampfia russelli Singh, Vahlkampfia ovis Schmidt, and Leptomyxa flabellata Goodey. Among the species isolated Naegleria sp., A. polyphaga, A. culbertsoni, and A. castellanii include strains, known to be pathogenic in humans.

Ultrastructure of cyst differentiation in parasitic protozoa.

Cysts represent a phase in the life cycle of biphasic parasitic protozoa that allow them to survive under adverse environmental conditions. Two events are required for the morphogical differentiation from trophozoite to cyst and from cyst to trophozoite: the encystation and excystation processes. In this paper, we present a review of the ultrastructure of the encystation and excystation processes in Entamoeba invadens, Acanthamoeba castellanii, and Giardia lamblia. The comparative electron microscopical observations of these events here reported provide a morphological background to better understand recent advances in the biochemistry and molecular biology of the differentiation phenomena in these microorganisms.

Ultrastructural study of encystation and excystation in Acanthamoeba castellanii.

Abstract. Encystation and excystation of Acanthamoeba castellanii were studied by transmission electron microscopy. The differentiation process was induced in asynchronous cultures grown axenically. Cytoplasmic vesicles containing a dense fibrous material very similar in appearance to the cyst wall were observed in trophozoites induced to encyst. When these trophozoites were incubated with calcofluor white m2r, fluorescence was observed in cytoplasmic vesicles, suggesting that the material contained in these vesicles corresponded to cyst wall precursors. Semithin cryosections of mature cysts with the same treatment showed fluorescence in the ectocyst and a less intense fluorescence in the endocyst, suggesting the presence of cellulose in both structures of the cyst wall. In mature cysts induced to excystation, small structures very similar to electron‐dense granules (EDG) previously described in other amoebae were frequently observed. The EDGs were either sparsely distributed in the cytoplasm or associated with the cytoplasmic face of the plasma membrane. Many of them were located near the ostiole. In advanced phases of excystation, endocytic activity was suggested by the formation of endocytic structures and the presence of vacuoles with fibrous content similar to that of the cyst wall. Electron‐dense granules in the process of dissolution were also observed in these vacuoles. Furthermore, the formation of a pseudopod suggests a displacement of the amoeba toward the ostiole.

Pathogenic Amoebae in Natural Thermal Waters of Three Resorts of Hidalgo, Mexico

In a search for free-living amoebae, seven water samples from three thermal water bathing resorts in Tecozautla, Hidalgo, were analyzed during December 1984. The samples were concentrated by filtration and centrifugation, and inoculated later on monoxenic and axenic media. The identification of the isolates was performed by morphology and isoelectric focusing of isoenzymes and total proteins. Thirty-three strains of free-living amoebae belonging to the genera Naegleria, Acanthamoeba, and Willaertia were isolated. Twenty of these strains belonged to the Naegleria genus, 16 of them were classified as Naegleria spp., and 2 were classified as Naegleria lovaniensis. Noteworthy was the finding of two pathogenic strains of the species Naegleria australiensis. N. australiensis and N. lovaniensis may be considered good indicator organisms, since they live in the same environmental conditions as N. fowleri, the agent of primary amoebic encephalitis (PAM). On the other hand, amoebae other than Naegleria were isolated and identified as Acathamoeba castellanii (two strains), and Acanthamoeba lugdunensis (one strain), which proved to be pathogenic when tested in mice. Nine more pathogenic strains of the genus Acanthamoeba spp. were isolated together with one strain of Willaertia magna, a thermophilic nonpathogenic amoeba. The chlorination and periodical surveillance of water resorts like the one studied is recommended, in order to prevent the appearance of more cases of PAM or other human diseases associated with pathogenic Acanthamoeba spp.

Seasonal distribution of air-borne pathogenic and free-living amoebae in Mexico City and its suburbs

A survey was carried out over a one-year period to isolate amoebae suspended in the air of Mexico City and its suburbs. Sampling stations were placed at the four cardinal points of the metropolitan area. Selective media were used to culture the amoebae isolated. Specialized taxonomic keys and physical and physiological tests were used for identification, and a statistical analysis was performed to determine the correlations between physico-chemical and biological parameters. 108 strains were isolated, of which 19 were pathogenic via intracerebral inoculation and 9 via intranasal inoculation. Species of the generaAcanthamoeba, Vahlkampfia andHartmannella were most abundant.Acanthamoeba polyphaga showed the highest abundance. Several times during the period of the study SO2 O3, CO, NO and NO2 exceeded the permissible levels established by the Mexican government. The ability of amoebae to form cysts and cyst size were important factors for their presence, survival, abundance and diversity in the atmosphere. The main source of air-borne amoebae was the soil. Factors that favored the incidence and diversity of the isolates were wind speed and direction, low relative humidity, generation of frequent dust-storms, resuspension of amoebae by vehicular traffic, proximity to garbage dumps and large extensions of bare soil. Soil cover was a factor associated with a reduction in the incidence and diversity of the aerial amoebae. This study demonstrates that there are viable cysts of amoebae in the atmosphere of Mexico City, that may have potential importance in the case of certain kinds of human allergies and diseases. Further research is needed to find out the aerial presence of viable cysts of obligatory, amphizoic or opportunistic amoebic parasites, and to clarify the qualitative and quantitative effects of the local meteorological and physico-chemical environment on the free-living amoebae present in the atmosphere.

SEASONAL DISTRIBUTION OF AIR-BORNE PROTOZOA IN MEXICO CITY AND ITS SUBURBS

A survey was carried out over a 1-yr period to isolate protozoa suspended in the air of Mexico City and its suburbs. Sampling stations were placed at four cardinal points of the metropolitan area. Selective media were used to culture the protozoa isolated. Specialized taxonomic keys were used for identification and a statistical analysis was performed to determine the correlations between physicochemical and biological parameters. 63 strains were isolated. Species of Kinetoplastida and Chrysomonadida were most abundant, namely species of the genera Bodo, Cercobodo, Monas and Helkesimastix. Amoeboflagellates found included Mastigamoeba and Tetramitus. The only ciliate isolated was Colpoda steini. The greatest number of strains was yielded by Bodo repens and Cercobodo radiatus. Several times during the period of the study SO2, O3, CO, NO, and NO2 exceeded the permissible levels established by the Mexican government. From the results of this study it was concluded that the ability of protozoa to form cysts and cyst size were important factors for their presence, survival, abundance and diversity in the atmosphere. The main source of air-borne protozoa was the soil. Factors that favored the incidence and diversity of the isolates were wind speed and direction, high relative humidity, generation of frequent dust-storms, resuspension of protozoa by vehicular traffic, proximity to garbage dumps and large extensions of bare soil, and temperature (the latter only when it did not favor the production of high levels of O3). Factors that were hostile to the aerial protozoa were industry and their contaminants, and high concentrations of O3 and SO2, especially in winter time when thermal inversions occurred. Soil cover was also associated with a reduction in the incidence and diversity of the aerial protozoa. This study demonstrates that there are viable cysts of protozoa in the atmosphere of Mexico City, that may have potential importance in the case of certain kinds of human allergies and diseases. Further research is needed to find out the aerial presence of viable cysts of obligatory, amphizoic or opportunistic protozoan parasites, and to clarify the qualitative and quantitative effects of the local meteorological and physico-chemical environment on the free-living protozoa present in the atmosphere.

Ultrastructural Study of the Encystation and Excystation Processes in Naegleria sp.

ABSTRACT. An important aspect of the biology of Naegleria sp. is the differentiation processes that occur during encystation and excystation. We studied these using both fluorescence and transmission electron microscopy techniques. In the initial stages of encystation, the cisternae of the endoplasmic reticulum became densely filled with a fibrillar material. Vesicles with a similar content that appeared to be derived from the cisternae were also observed in close contact with the plasma membrane. As encystation progressed, the fibrillar material became localized on the surface of the amoeba. An irregular compaction was observed in some areas of the cyst wall, which contained thin extensions of the cyst wall fibrillar material. Completely formed cysts had two to three ostioles, each sealed by an operculum. The operculum contained two areas in which a differential compaction of the fibrillar structure was observed. When excystation was induced, small dense granules (DGs), which were in close contact with fibrillar material were observed in the cyst cytoplasm and in the peritrophic space. During excystation, the more compact component of the operculum moves to enable the pseudopod of the emerging trophozoite to penetrate the ostiole. Vacuoles containing a fibrillar material, probably derived from the cyst wall, were observed in the cytoplasm of the pseudopodia. Our results provide a platform for further studies using biochemical markers to investigate the origin of the cyst wall as well as the role of DGs during excystation in Naegleria.

Amoebae in a waste stabilization pond system in Mexico

A protozoological survey was performed to analyze the organisms of the subphylum Sarcodina Schmarda, present in waste stabilization ponds located at Santo Tomás Atzingo, México, from March to December 1981. The amoebae isolated were identified and counted. Several physicochemical parameters were also determined and correlated with the biological data.Thirteen amoebic species were isolated and identified: one definite pathogen, Entamoeba histolytica Schaudinn; three species that have shown pathogenic strains for humans: Acanthamoeba polyphaga Page, Acanthamoeba castellanii Douglas, and Naegleria sp. (Alexeieff) Calkins; two opportunist species: Entamoeba coli (Grassi) Casagrandi and Barbagallo, and Iodamoeba bühschlii (Von Prowazek) Dobell; and seven free-living amoebae: Acanthamoeba astronyxis Ray and Hayes, Amoeba proteus (Pallas) Leidy, Hartmannella exundans Page, Hartmannella vermiformis Page, Pelomyxa palustris Leidy, Vahlkampfia avara Page, and Vahlkampfia russelli Singh.A clear correlation between the number of amoebae and the temperature and alkalinity of the system was found. The removal capacity of the ponds for E. histolytica varied from 30 to 100% during the survey. The amoebae showed a succession in space and time in the system studied.

An investigation of the pathogenic and non-pathogenic free-living amoebae in an activated-sludge plant

Pathogenic and non-pathogenic free-living amoebae from an activated-sludge plant were investigated. The greatest species diversity occurred in the raw, settled sewage and at the beginning of the aeration tank, whilst the lowest number of species occurred in the final sedimentation tanks. Thirteen species of free-living amoebae representing eleven genera were isolated; these included three species of Acanthamoeba (A. castellanii, A. culbertsoni and A. polyphaga), all of wich were pathogenic to mice with varying degrees of virulence. The implications for the public health risk of the presence of pathogenic free-living amoebae in activated-sludge plants are discussed.