The free-living amoeboflagellate genus Naegleria includes one pathogenic and two potentially pathogenic species (Naegleria fowleri, Naegleria italica, and Naegleria australiensis) plus numerous benign organisms. Monitoring of bathing water, water supplies, and cooling systems for these pathogens requires a timely and reliable method for identification, but current DNA sequence-based methods identify only N. fowleri or require full sequencing to identify other species in the genus. A novel closed-tube method for distinguishing thermophilic Naegleria species is presented, using a single primer set and the DNA intercalating dye SYTO9 for real-time PCR and melting-curve analysis of the 5.8S ribosomal DNA gene and flanking noncoding spacers (ITS1, ITS2). Collection of DNA melting data at close temperature intervals produces highly informative melting curves with one or more recognizable melting peaks, readily distinguished for seven Naegleria species and the related Willaertia magna. Advantages over other methods used to identify these organisms include its comprehensiveness (encompassing all species tested to date), simplicity (no electrophoresis required to verify the product), and sensitivity (unambiguous identification from DNA equivalent to one cell). This approach should be applicable to a wide range of microorganisms of medical importance.

This is the first extensive study of soil protozoa of arid lands. Twenty-six samples from litters, soils, termitaria, and a cyanobacterial crust, collected from central and south Australian arid lands, were analyzed for numbers and species of gymnamoebae, ciliates, and testacea. Amoebae ranged from 1,000-5,000/g of material, and were two orders of magnitude more abundant than ciliates. Both groups increased in abundance and species richness from bare soils through spinifex to mulga to chenopod vegetations. Testacea ranged 900-5,000/g with similar species richness throughout vegetations, but reached 11,900/g with a doubling of species in a refugium in Kings Canyon. The most prevalent species of amoebae, ciliates, and testacea were taxa associated with ephemeral and disturbed habitats (r-selection). The cyanobacterial crust might be considered a micro-refugium because it contained a number of non-encysting protozoa, including Thecamoeba sp. and Nassula picta, feeding on cyanobacterial filaments. The numbers and species richness of protozoa under shrubs were greater than in bare soils, supporting the resource island hypothesis that desert plants create soil heterogeneity by localizing soil fertility under their canopies.

Cryptosporidium parvum are protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring within Cryptosporidium sporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37 degrees C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.