Una Ryan

One of the most challenging issues in food safety is the detection of foodborne pathogens. Since the infectious dose of many pathogens is low, the sensitivity of the diagnostic tool becomes important. Traditionally, the detection and analysis of foodborne pathogens have relied on light or electron microscopy and culture methods. Conventional techniques such as microscopy lack sensitivity, are labor intensive, and require well-trained microscopists for accurate identification and interpretation, particularly for pathogens that are morphologically similar or very small in size or present in very low numbers. Special stains are required for the diagnosis of some pathogens. In addition, the diagnostic skills of microscopists can vary greatly from laboratory to laboratory resulting in some infections being misdiagnosed or missed completely. Furthermore, light microscopy can have a low sensitivity of detection, which is particularly relevant, considering that, for some pathogens, just a few individuals within a sample may represent an infectious dose.

Cryptosporidium parasites belong to the phylum Apicomplexa and possess features of both the coccidia and gregarines. Currently, 25 species of Cryptosporidium are recognized in fish, amphibians, reptiles, birds and mammals. All 25 species have been confirmed by morphological, biological, and molecular data. Cryptosporidium duismarci and C. scophthalmi lack sufficient biological and/or molecular data to be considered valid species. In addition to the named species, more than 40 genotypes from various vertebrate hosts have been described. For these genotypes to receive taxonomic status, sufficient morphological, biological, and molecular data are required and names must comply with the rules of the International Code for Zoological Nomenclature (ICZN). A different interpretation of the ICZN led to the proposal that Cryptosporidium parvum be renamed as Cryptosporidium pestis and that C. parvum be retained for C. tyzzeri. However, this proposal violates the guiding ICZN principle of maintaining taxonomic stability and avoiding confusion. In addition, C. pestis lacks a full taxonomic description and therefore is not a valid species. The taxonomic status of Cryptosporidium spp. is rapidly evolving and many genotypes are likely to be formally described as species in the future.

Cryptosporidium is an important enteric parasite and has been identified as the cause of numerous waterborne, foodborne, and day-care outbreaks of diarrheal disease worldwide. Ill. The oocyst is the environmentally stable stage and is able to survive and penetrate routine wastewater treatment and is resistant to inactivation by commonly used drinking water disinfectants. Currently, cryptosporidiosis represents the major public health concern of water utilities in developed nations and has been responsible for 50.8% (165/325) of all water-associated outbreaks of parasitic protozoan disease reported in North American and Europe.

Cryptosporidium has been reported in a wide variety of hosts, with C. parvum and C. hominis being responsible for most human infections. Until recently, it has been assumed that farm animals and wild animals are important zoonotic reservoirs for human cryptosporidiosis. However, recent molecular analysis has revealed a wide range of Cryptosporidium species and genotypes infecting both domestic and wild animals, and the epidemiology of cryptosporidiosis is clearly more complicated than was previously thought.

Because of the ability of Cryptosporidium species to infect humans and a wide variety of animals, and because of the ubiquitous presence of cryptosporidium oocysis in the environment, humans can acquire Cryptosporidium infections through several transmission routes Hunter and Nichols, 2002; Chapter 4). These include direct person-to-person or animal-to-person transmission and indirect waterborne and foodborne transmission, and the parasites can be of anthroponotic or zoonotic origin. The role of each transmission route in endemic areas, however, is frequently unclear because of the expensive nature of epidemiologic investigations and the inability to differentiate Cryptosporidium species by conventional microscopy. Molecular tools have been developed to detect and differentiate Cryptosporidium at the species/genotype and subtype levels (Xiao and Ryan, 2004; Caccio, 2005). The use of these tools has made significant contributions to our understanding of the biology and epidemiology of Cryptosporidium species. This includes better knowledge of the species structure and population genetics of Cryptosporidium, the roles of various transmission mutes in cryptosporidiosis epidemiology, and the significance of parasite genetics in pathogenesis and clinical presentations. These recent developments have enabled researchers to make more accurate risk assessment of environmental and drinking water contamination, and have helped health officials to better educate the public on risk factors invoked in the acquisition of cryptosporidiosis in vulnerable populations.

Techniques based on nucleic acid amplification have proven to be essential for the detection and epidemiological tracking of members of the genus Cryptosporidium. This gastrointestinal protozoan parasite cannot be routinely cultivated and it has an extremely low infectious dose, possibly below 100 oocysts. As Cryptosporidium is an important pathogen, particularly in immuno-compromised hosts, there is a pressing need to employ sensitive and discriminatory systems to monitor the organism. A number of fairly standard target genes have been assessed as detection targets, including 18S rRNA, microsatellites, and heat-shock (stress) proteins. As our knowledge of the biology of the organism increases, and as the full genome information becomes available, the choice of target may change. Genes encoding parasite-specific surface proteins (gp60, TRAP-C2, COWP) have already been examined. Much of the effort expended in molecular diagnostics of Cryptosporidium has been directed toward developing robust nucleic acid extraction methods. These are vital in order to recover amplifiable DNA from environments where small numbers of oocysts, often fewer than 100, may exist. Methodology based on adaptation of commercial kits has been developed and successfully employed to recover amplifiable DNA directly from water, food (particularly seafood), and fecal samples.

This chapter presents an overview on the successful cultivation of Cryptosporidium. The successful cultivation reveals previously undescribed gregarine-like developmental stage. s. Cryptosporidium shows peculiarities that separate it from other coccidia, such as two morphofunctional oocysts, a multi-membranous feeder organelle, and endogenous development in the microvilli of epithelial surfaces. Cryptosporidium has a closer affinity with the gregarines than with the coccidian. This chapter presents a study in which at least two developmental stages in Cryptosporidium are found to be similar to those of some gregarines. These are an extracellular trophozoite/gamont-like stage and a gametocyst-like stage. Both stages were detected in vitro and in vivo. Furthermore, surface association of gamont-like stages as well as oocyst formation by budding from gametocyst stages was observed in this study.