Detection And Enumeration Methodologies

Methodologies for the identification and isolation of cryptosporidium in water have been thoroughly studied. The EPA Method 1623 [61,62] is based on the recovery of cryptosporidium oocysts and giardia cysts by filtration/IMS (immunomagnetic separation)/FA (fluorescent antibody) (EPA-821-R-99-006) of up to 101 of water. Filtration can be performed using either the Pall Gelman HV Envirochek® capsule or the IDEXX Filta-MaxTM filter. Cysts or oocysts are then captured by IMS using Dynabeads® GC-Combo (Dynal, Inc.) or Aureon CG (Aureon Biosystems) kits. Once the oocysts are recovered, they are identified using immunofluorescent assays from Merifluor® G/C (Meridian Diagnostics, Inc.), Aqua-GloTM G/C Direct (Waterborne, Inc.), or Crypto-GloTM (BioTechFrontier, Inc.). Recovery efficiency for Cryptosporidium parvum is 60 to 80%. Heat incubation of IMS-tagged oocysts resulted in recoveries of 71 and 51% and DAPI confirmation rates in reagent and river water of 93 and 73%, respectively [63]. Method 1623 has several limitations and interferences. IMS can be affected by water turbidity and the presence of silica, clay, humic acids, other organisms, etc. The presence of iron and pH will also affect oocyst recovery.

Electrochemiluminescence (ECL) technology has been used to identify cryptosporidium in environmental water samples of up to 10,000 nephelo-metric turbidity units [64].

It is also important to determine whether the parasites are viable and of public health relevance. Molecular assays will aid in speciation and subtyping of the parasites. These include polymerase chain reaction (PCR), reverse transcription (RT)-PCR, nested PCR, and an isothermal amplification nucleic acid sequence-based amplification (NASBA) method [65-70]. After isolation of the parasites, extraction of the oocyst DNA is of critical importance. Oocysts can be broken by boiling, mechanical disruption with glass beads, digestive enzymes (proteinase-K, lysozyme) with 10% SDS, freeze/thaw, microwave, sonication, and commercial kits (DNA and RNA) or automated systems (contamination free) [71-73].

In water samples, various Cryptosporidium parvum mRNAs have been used as molecular targets for detection [65]. The mRNA coding of C. parvum for hsp70 was amplified using NASBA methodology with a detection limit of 80fmol amplicon/test. [74].

Because mRNA denatures quickly, oocyst viability can be determined using RT-PCR for cryptosporidium using the hsp70 and the p-tubulin genes. An electrochemical enzyme-linked immobilized DNA-hybridization assay using the C. parvum hsp70 mRNA could distinguish dead from live oocysts. No cross-reactivity was observed with other bacterial and parasitic organisms, including Cryptosporidium muris [75].

In vitro cultivation recognizes parasites that are both viable and have the ability to penetrate and replicate within host cells. Infectivity can be determined using animal models, but C. hominis, which is the anthroponotic species, is host specific and is not infectious in neonatal mouse models.

Some of these methods have been used in food matrices. An IMS-PCR assay was able to detect <10 C. parvum oocysts in milk [76].

A laser scanning cytometry method (ChemScanRDI), coupled with immunofluorescence detection with differential interference contrast (DIC) confirmation, has also been evaluated and compared with manual microscopic enumeration of cryptosporidium oocysts. The recovery rate was 50% at seeding levels from 30 to 230 oocysts. Laser scanning cytometry does eliminate the low sample throughput, operator subjectivity, and operator fatigue using conventional microscopy [77].

Although these methodologies have been described for environmental waters, they have not been fully validated in foods. The wide variety of produce and foods that could potentially be involved in parasite transmission makes selecting a unique method for isolation difficult. Detection using molecular diagnostic assays is also challenging because of the presence of inhibitors that could mask the presence of these parasites in foods.

Immunoassays have been developed for the use of cryptosporidium identification in water samples. An indirect immunofluorescent assay has also proven to be useful in food matrices [23,78].

Most of the purification techniques that work for cryptosporidium have proven to be effective in purifying cyclospora oocysts (Ortega, personal communication). Sucrose and cesium chloride gradients used for cryptospori-dium can be used for cyclospora. Water filtration systems have also proven to concentrate cyclospora from water sources. To date, monoclonal antibodies to cyclospora have not been produced. This is not only because of the limited sources of oocysts, but also because the cell wall has poor antigenic properties. One useful approach has been to use magnetic beads coated with the lectin WGA [79].

Various methodologies have been described to identify cyclospora using conventional clinical assays. When environmental samples are examined, autofluorescence can prove useful, although this is not a specific assay. A PCR method, initially designed for clinical samples, has worked well with food matrices; however, other Eimeria spp. also had the same amplification product as cyclospora [80]. A restriction fragment length polymorphism using the Mnl 1 enzyme could differentiate between cyclospora and eimeria. The biggest challenges when using PCR for cyclospora are the methodologies used to extract DNA from the low number of oocysts likely to be encountered, and how to control for the presence of PCR inhibitors. Various methodologies, including chelating matrices and freeze/thaw cycles, FTA membranes, and DNA extraction kits, have been described [72,80]. The use of an extraction-free, filter-based protocol (FTA) to prepare DNA templates for use in PCR to identify C. cayetanensis and C. parvum oocysts and microsporidia spores has been described. As few as 10 to 30 C. cayetanensis oocysts per 100 g of fresh raspberries could be detected [72].

To control for PCR inhibitors, addition of BSA or milk has improved the sensitivity of the assay. The PCR assay in raspberries, basil, and mesclun lettuce could detect 40 or fewer oocysts per 100 g of raspberries or basil, but had a detection limit of around 1000 per 100 g in mesclun lettuce [72,81]. Real-time PCR can also detect DNA specifically from as few as 1 oocyst of C. cayetanensis per 5 ^ reaction volume [82].

Determining the viability of cyclospora oocysts has proven to be very difficult. To date, there are no susceptible animal models or in vitro cultivation methods. Sporulation rates have been used to determine if a particular treatment has affected the oocyst viability. This, however, may not have any bearing on oocyst infectivity. Electrorotation has been used as a method to determine oocyst viability [83]. This method needs to be validated when an in vivo or in vitro system for cyclospora becomes available.

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