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The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 x 10(6) oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum "bovine" genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium "marsupial" genotype I or "marsupial" genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.

Molecular characterisation of Cryptosporidium oocysts isolated from faeces collected from eastern grey kangaroos Macropus giganteus inhabiting an Australian water catchment revealed that this host was susceptible to three types of Cryptosporidium. Nucleotide sequence analysis of the 18S rDNA, Cryptosporidium oocyst wall protein (COWP) and a 70kDa heat shock protein (HSP70) identified an isolate identical to the described Cryptosporidium 'marsupial' genotype. A second isolate had less than 0.5% variation, compared to the described Cryptosporidium 'marsupial' genotype, within the sequences of the 18S rDNA, COWP and HSP70 and 10% variation in the internal transcribed spacer 1 (ITS1). Multilocus analysis of the third Cryptosporidium revealed a novel genotype that had a degree of genetic variation, at the four loci characterised, which was greater than or equivalent to that used to discriminate between currently recognised Cryptosporidium species. These findings have increased our current understanding on the molecular epidemiology of Cryptosporidium in Australian wildlife and have provided information on the types of Cryptosporidium marsupials may shed into the environment.

A combined immunomagnetic separation (IMS) and flow cytometry (FC) technique was developed for the sensitive detection of Cryptosporidium in faecal samples. The IMS/FC technique was found to be approximately 50-fold more sensitive than formol-ether concentration, which is commonly used for Cryptosporidium epidemiological investigations. Of 31 faecal samples from captive animals 16 were found to contain Cryptosporidium oocysts when analysed using the IMS/FC compared to four when using formol-ether concentration (FEC). In a wild population of eastern grey kangaroos Macropus giganteus 66.3% of infected animals were shedding <500oocysts/gfaeces when analysed using IMS/FC. This is below the detection limit for the FEC method. The dispersal of Cryptosporidium in host populations is aggregated, with many individuals shedding low numbers of oocysts and few individuals shedding numbers of oocysts sufficiently high to be detected by FEC. This research demonstrates that the prevalence and oocyst shedding intensity of Cryptosporidium in animal populations will be significantly underestimated using standard detection methods.

Large numbers of microbiological samples are analysed annually using traditional culture-based techniques. These techniques take hours to days to yield a result, are tedious and are not suitable for non-culturable microorganisms. Further, culture-based techniques do not provide real-time information on the physiological status of the organism in situ which is important in the industrial manufacture of many microbial products. Flow cytometry offers the prospect of real-time microbial analysis of individual microorganisms, without dependency on microbial culture. However, flow cytometry has not been extensively used as a tool for routine microbial analysis. This has been mainly due to the high cost and complexity of instrumentation, the need for trained flow cytometrists and the lack of assay kits with appropriate biological reagents for specific applications. Many modern instruments are now relatively simple to operate, due to improvements in the user-interface, and no longer need a specialist operator. However, most cytometers are still reliant on analogue technology first developed 20-30 years ago. The incorporation of modern, solid state opto-electronics combined with micro-fabrication and digital signal processing technology offers the prospect of simple to use, low cost and robust instruments suitable for microbial analyses. Advances are being made in the development of a range of biological reagents and these are now being formulated into simple to use kits for microbiological applications. Currently, these kits are largely restricted to simple analyses, for example to assay for total or viable numbers of microorganisms present. However, technologies are available to selectively label specific types of microorganisms. For example, fluorescent antibodies can be used to label microorganisms according to expression of particular antigens, fluorescent in situ hybridisation to label according to phylogeny and fluorogenic enzymatic substrates to label according to expression of specific enzyme activities. Reagents are also available that stain viruses sufficiently brightly to enable their direct detection in environments such as sea water. Microorganisms need to be detected in a variety of different matrices (e.g., water, mud, food, and beverages) and these matrices may be highly variable in nature (e.g., tap water compared to river water). Many matrices have high background autofluorescence (e.g., algae and minerals in water samples) or may bind non-specifically to the fluorescent biological reagents used (e.g., protein micelles in milk). Formulation of biological reagents and sample pre-treatments are critical to the development of suitable microbiological assays. Here, developments in instrumentation and biological reagents for microbiological applications are reviewed with specific examples from environmental or industrial microbiology. The broader considerations for the development of microbial assays for flow cytometry are also considered.

A simple method is described for the evaluation and quality control of Cryptosporidium-specific antibodies used in monitoring environmental water samples. Purified oocysts were fluorescently labelled with a test antibody at the appropriate concentration. Labelled oocysts were analysed using flow cytometry and a region was defined on a bivariate dotplot of fluorescence versus light scatter that enclosed all oocysts. Concentrates of environmental water samples that did not contain oocysts were then incubated with the test antibody and analysed using flow cytometry. The number of particles that appeared in the region defined for oocysts was recorded and was a measure of non-specific binding. The technique provides a simple, rapid and quantitative tool for both evaluating the binding specificity of test antibodies and optimizing sample staining conditions.

In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.