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Abstract Background DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. Results The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro. Conclusion Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons.

Cryptosporidium are ubiquitous and significant enteropathogens of all classes of vertebrates and a major cause of human morbidity and mortality worldwide. Of the 24 recognized species, the zoonotic Cryptosporidium parvum and the host-specific Cryptosporidium hominis cause the majority of cases of human cryptosporidiosis. Here, we report on structural and transcriptional variability between C. parvum and C. hominis at the MIC1 locus, which encodes a microneme localized thrombospondin-like domain containing protein previously demonstrated to be critical for host cell infection by C. parvum. We demonstrate, using reverse transcription quantitative PCR with the aid of genomic data from the Eu Path DB site, that the transcribed product in C. hominis is both truncated and significantly down-regulated in the sporozoite. We hypothesize that Cp MIC1 may be a genetic factor involved in facilitating the wider host range of C. parvum in comparison with the specific host range of C. hominis. Furthermore, we show that the presence of a microsatellite ( ML-2) within the C. parvum MIC-1 locus enables the development of a PCR marker that can rapidly distinguish the zoonotic C. parvum from C. hominis and other significant human infectious Cryptosporidium species due to reproducible PCR slippage across the ML-2 microsatellite. Additionally, we demonstrate that this locus is tightly linked to the GP60 locus, a locus commonly used in the genetic characterization of C. parvum and C. hominis isolates. This marker should provide a robust and additional tool to aid in the rapid identification of C. parvum from other Cryptosporidium species. [ABSTRACT FROM AUTHOR]

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.

The taxonomy, life cycle patterns and zoonotic potential of Giardia infecting mammals and birds have been poorly understood and controversial for many years. The development of molecular tools for characterising isolates of Giardia directly from faeces or environmental samples has made an enormous contribution to resolving these issues. It is now clear that the G. duodenalis morphological group is a species complex comprising a series of what appear to be largely host-adapted species, and at least two zoonotic species for which humans are the major host, but which are also capable of infecting other mammals. It is proposed that this new information be reflected in the redesignation of several species of Giardia described previously. The molecular epidemiological tools that are now available need to be applied in different endemic foci of Giardia transmission, as well as in outbreak situations, in order to understand better the frequency of zoonotic transmission as well as to develop more effective approaches to controlling giardiasis.

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.

A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential.

Isolates of Cryptosporidium were characterized using nucleotide sequence analysis of the 18S rRNA and dihydrofolate reductase genes and also random-amplified polymorphic DNA analysis. Phylogenetic analysis confirmed the validity of the species of Cryptosporidium examined in this study such as Cryptospordium muris and Cryptosporidium baileyi, and also reinforced evidence from numerous researchers worldwide suggesting that Cryptosporidium parvum is not a single uniform species. The data obtained provided strong support for the validity of Cryptosporidium felis. Evidence suggests that the newly identified marsupial and pig genotypes may also be distinct and valid species, but biological studies are required for confirmation.