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Cryptosporidiosis is predominantly a gastrointestinal disease of humans and other animals, caused by various species of protozoan parasites representing the genus Cryptosporidium. This disease, transmitted mainly via the faecal-oral route (in water or food), is of major socioeconomic importance worldwide. The diagnosis and genetic characterization of the different species and population variants (usually recognised as "genotypes" or "subgenotypes") of Cryptosporidium is central to the prevention, surveillance and control of cryptosporidiosis, particularly given that there is presently no broadly applicable treatment regimen for this disease. Although traditional phenotypic techniques have had major limitations in the specific diagnosis of cryptosporidiosis, there have been major advances in the development of molecular analytical and diagnostic tools. This article provides a concise account of Cryptosporidium and cryptosporidiosis, and focuses mainly on recent advances in nucleic acid-based approaches for the diagnosis of cryptosporidiosis and analysis of genetic variation within and among species of Cryptosporidium. These advances represent a significant step toward an improved understanding of the epidemiology as well as the prevention and control of cryptosporidiosis.

Sound application of molecular epidemiological principles requires working knowledge of both molecular biological and epidemiological methods. Molecular tools have become an increasingly important part of studying the epidemiology of infectious agents. Molecular tools have allowed the aetiological agent within a population to be diagnosed with a greater degree of efficiency and accuracy than conventional diagnostic tools. They have increased the understanding of the pathogenicity, virulence, and host-parasite relationships of the aetiological agent, provided information on the genetic structure and taxonomy of the parasite and allowed the zoonotic potential of previously unidentified agents to be determined. This review describes the concept of epidemiology and proper study design, describes the array of currently available molecular biological tools and provides examples of studies that have integrated both disciplines to successfully unravel zoonotic relationships that would otherwise be impossible utilising conventional diagnostic tools. The current limitations of applying these tools, including cautions that need to be addressed during their application are also discussed.

The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6-7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.

Giardia and Cryptosporidium are enteric protozoan parasites that infect a wide range of vertebrate hosts. Both are transmitted either by direct faecal/oral contact or by the ingestion of contaminated food or water. The discovery of morphologically similar organisms infecting humans and a variety of mammals and birds has led to the proposal that both Cryptosporidium and Giardia are zoonotic (i.e. transmitted in nature between humans and animals). Transmission between humans and animals has been supported by cross-infection studies. However, closer examination of many of these studies reveals limitations in the methodologies utilised. More recent molecular genetic studies have demonstrated considerable genetic diversity among isolates of the same species of Giardia and Cryptosporidium, suggesting that these species are in fact species complexes and that some of these novel species may be host-specific. This paper will critically examine the evidence for the zoonotic transmission of these parasites.

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

The discipline of systematics plays a central role in all branches of biology. In today's technology-orientated research world, it is important to realise the continuing value of systematics, the basic tenet of which is to combine diverse types of data to produce classifications that reflect the natural history of living organisms. Accurate classification systems are crucial in the field of parasitology, not only because they provide the means to identify species and strains of parasites, but also because they provide a framework around which a parasite's biology can be studied. The construction of such a classification system is often hampered by the parasite's biology, which may preclude the application of traditional techniques or concepts (such as morphological differentiation or the biological species concept) to delineate species. It is often the case that these difficulties can be overcome by the use of molecular systematic techniques. In this paper, it is proposed that a detailed understanding of the phylogeny of a group of organisms can be used as a basis to examine other aspects of their systematics. This is illustrated using the protozoan parasite Giardia intestinalis. Data gathered using the complementary techniques of allozyme electrophoresis and nucleotide sequencing have been used to infer the phylogenetic relationships of G. intestinalis isolated from various host species. The results, supported by biological data, suggest that G. intestinalis is a species-complex. As we move towards the year 2000, molecular systematics will play an increasingly important role in elucidating host-parasite relationships. However, its use as a taxonomic tool will require a general acceptance by parasitologists and the adoption of formal procedures to allow the description of new species by these methods. The aim of this approach is not to dismiss traditional methods, but to use them in combination with contemporary methods in the true spirit of the discipline of systematics.

The extent of intra-specific genetic variation between isolates of Giardia muris was assessed by allozyme electrophoresis. Additionally, the levels of allozymic variation detected within G. muris were compared with those observed between members of the two major assemblages of the morphologically distinct species Giardia intestinalis. Four isolates of G. muris were analysed. Three (Ad-120, -150, -151) were isolated from mice in Australia, while the fourth (R-T) was isolated from a golden hamster in North America. The 11 isolates of G. intestinalis (Ad-1, -12, -2, -62, representing genetic Groups I and II of Assemblage A and BAH-12, BRIS/87/HEPU/694, Ad-19, -22, -28, -45, -52, representing genetic Groups III and IV of Assemblage B) were from humans in Australia. Intra-specific genetic variation was detected between G. muris isolates at four of the 23 enzyme loci examined. Similar levels of variation were found within the genetic groups that comprise Assemblages A and B of G. intestinalis. These levels of intra-specific variation are similar to those observed within other morphologically-distinct species of protozoan parasites. We suggest that the magnitude of the genetic differences detected within G. muris provides an indication of the range of genetic variation within other species of Giardia and that this can be used as a model to delineate morphologically similar but genetically distinct (cryptic) species within this genus.

An understanding of the epidemiology of a disease (i.e. its aetiology, transmission patterns) is crucial for the development and implementation of effective management practices. This requires sound epidemiological data. It is therefore important that scientists understand the assumptions and limitations of the methods used to gather such data. The aim of this paper is to discuss some of the assumptions and limitations of PCR-based methods used in studies of epidemiology. Since its development, PCR has had a major impact in the biological sciences. The ability to selectively amplify a specific region of the genome from a small amount of DNA makes this technique particularly useful as a diagnostic tool. A variety of PCR-based methods are available which can be used to identify strains and species of parasites. Some of these methods, such as random amplification of polymorphic DNA, have intrinsic properties which can limit their application. Other methods, such as PCR-restriction fragment length polymorphism, require the availability of a sound taxonomic or genetic framework for the development of any diagnostic system for a particular organism. The problems encountered developing diagnostic probes in the absence of such a framework will be discussed using Giardia intestinalis as an example.