1. Materials and Equipment
2. Reagents
3. Optional Reagents, Supplies, and Equipment
4. Wash Procedure for Fresh Produce
   1. Isolation of Cyclospora from Fresh Produce Washes
   2. Isolation of Cryptosporidium spp. from Fresh Produce Washes
5. Isolation of Parasitic Contaminants from Juices, Cider, and Milk
   1. Isolation of Cyclospora from Juices, Cider, and Milk
   2. Isolation of Cryptosporidium spp. from Juices, Cider, and Milk
6. Isolation of Parasitic Contaminants from Large Volumes of Water
7. Slide Preparation and Microscopic Analysis-Cyclospora cayetanensis
   1. Sample Preparation
   2. Microscopy
8. Slide Preparation and Microscopic Analysis-Cryptosporidium spp.
9. PCR Analysis
   1. DNA Primers
   2. General Sample Preparations for Primary PCR Amplification
   3. Conventional Multiplex PCR Amplification for the Differential Identification of Cyclospora and Eimeria spp.
   4. OPTIONAL-Real Time Multiplex PCR Amplification for the Differential Identification of Cyclospora and Eimeria spp. using the Roche LightCycler^® System
   5. Nested PCR Amplification for the Differential Identification of Cryptosporidium spp.
   6. Agarose Gel Electrophoresis
   7. Restriction Fragment Length Polymorphism (RFLP) Analysis of Cryptosporidium spp. Nested PCR Amplicons to Determine the Presence and Differentiation of C. hominis and C. parvum

References

List of Tables

• Table 1: Parameters for Epifluorescence Microscopy
• Table 2: DNA Primer Sequences for Cyclospora-specific PCR Amplification
• Table 3:  DNA Primer Sequences for Cryptosporidium Genus-specific PCR Amplification
• Table 4: General PCR Conditions for Primary PCR Amplification
• Table 5: Controls for PCR Amplifications
• Table 6: PCR Thermal Cycling Parameters for Cyclospora and Eimeria spp. Detection
• Table 7: PCR Thermal Cycling Parameters for Cryptosporidium spp.
• Table 8: Assay Conditions for the Conventional Nested Multiplex PCR Amplification of Cyclospora and Eimeria spp.
• Table 9: Conditions for the Real-time Nested Multiplex PCR Amplification of Cyclospora and Eimeria spp.
• Table 10: LightCycler^® Thermal Cycling Parameters for Real-time Multiplex PCR Amplification of Cyclospora and Eimeria spp.
• Table 11: Expected Results for Real-time Multiplex PCR Amplification of Cyclospora and Eimeria spp. by Melting Curve Analysis
• Table 12: Assay Conditions for the Nested Amplification of Cryptosporidium spp.
• Table 13: Evaluation of nested PCR Amplification and RFLP Analysis to Differentiate Cryptosporidium spp.

1. Materials and Equipment

1. BagPage^®+ filter bags (400 ml) and Bag Clips (Interscience, St Nom, France)
2. Envirochek™ sampling capsule, 1 µm nominal (Pall Gelman Laboratory)
3. Rocker platform
4. Rotary shaker
5. Rotating wheel
6. 150 ml analytical filter units (Nalgene, Cat No. 130-4045)
7. 25 mm disposable filter funnels (Whatman Biosciences)
8. Dynal L10 tubes Prod. No. 740-03)
9. Dynal MPC^®-1 (Prod. No 120.01)
10. Dynal MPC^®-S (Prod. No 120.20)
11. Vacuum manifold (Vac-Man® Laboratory Vacuum Manifold 20-sample capacity; Promega)
12. FTA Filters, Classic Card of 960circle pre-printed card formats (Whatman Biosciences)
13. Photo laminating sheets (Scotch^®)
14. Conical 250 ml centrifuge tubes
15. Sorvall RT7 Plus refrigerated centrifuge or equivalent (to centrifuge 250 ml conical centrifuge tubes)
16. Epi-illuminated Fluorescence Microscope-equipped with the following: UV 1A filter block (excitation Filter, EX 365/10; Dichroic mirror, DM 440; Barrier Filter, BA-400; or equivalent. Optics for differential interference contrast (DIC). For Cryptosporidium spp.: appropriate filter for viewing fluorescein isothiocyanate (FITC) conjugated oocysts. (Table 1)
17. Glass microscope slides and cover slips
18. Blotting paper
19. Heating block for incubation at 56°C
20. Single punch (6 mm diameter) hole puncher
21. Thin-walled 0.65 ml PCR tube (PGC)
22. ART pipette tips (Molecular BioProducts)
23. PTC-200 DNA Engine (MJ Research) or comparable thermal cycler.
24. Horizontal gel electrophoresis apparatus and power supply.
25. Polaroid camera or digital imaging system to capture ethidium bromide-stained gels
26. Polaroid Type 667 film
27. UV Transilluminator

2. Reagents

1. Water
   1. Deionized water (for washing produce [dH₂O])
   2. Sterile deionized water (for PCR procedure)
2. Envirochek™ elution buffer (0.01M Tris, pH 7.4 ; 0.001 M EDTA; 1% SDS)
3. Silicone vacuum grease
4. Albumin, bovine (BSA) (Sigma, A-7030)
5. Celite (Sigma, C-8656)
6. Polyvinyl polypyrrolidone (PVPP) (Sigma, P-6755)
7. NET buffer: 0.1 M Tris, pH 8.0, 0.15M NaCl, 0.001M EDTA
8. NET-BSA buffer-NET buffer containing 1% (w/v) BSA
9. 20% celite in NET-BSA buffer (w/v)
10. 10% PVPP (w/v) in dH₂O
11. Dynabeads anti-Cryptosporidium Kit (Dynal Biotech Inc, USA)
12. Hydrofluor Combo Detection Kit for Cryptosporidium and Giardia (Strategic Diagnostics, Inc.)
13. 0.1 N HCl
14. 0.1 N NaOH
15. Immersion oil
16. Clear fingernail polish, slide compound, paraffin wax or equivalent
17. FTA Purification Buffer (Whatman Biosciences)
18. FTA filter wash buffer: 0.01 M Tris, Ph 8.0; 0.1 mM EDTA
19. DNA Primers-See Table 1 in PCR Section
20. HotStartTaq™ Master Mix Kit (Qiagen)
21. 0.5x Tris-acetate-EDTA buffer (0.5x TAE)
22. Molecular biology-grade agarose (BioRad)
23. Ethidium bromide
24. 6x gel loading solution
25. 100 bp and 25 bp DNA ladders (Invitrogen)
26. Vsp I restriction endonuclease (Promega)
27. Dra II restriction endonuclease (Hoffman-La Roche)
28. NuSieve® 3:1 agarose (Biowhitaker Molecular Applications)

3. Optional Reagents, Supplies, and Equipment

1. LightCycler^®-FastStart DNA Master SYBR Green I kit (Roche Diagnostics)
2. LightCycler^® Capillaries (Roche Diagnostics)
3. LightCycler^® System (Roche Diagnostics)

4. Wash Procedure for Fresh Produce

This procedure can be used to analyze for potential contaminants on fresh leafy produce (lettuce, herbs, etc) and berries (e.g. raspberries) and may be applicable to other fresh produce.

1. Place produce to be analyzed (10-25 g of fresh leafy produce or 50g of fresh berries) in a BagPage^®+ filter bag, add 100 ml dH₂O and seal with Bag Clip.
2. Place on rocker platform and gently agitate for 30 minutes at room temperature, inverting the bag after 15 minutes.
3. Decant supernatant from the BagPage^®+ filter bag into clean 50 ml conical centrifuge tubes and centrifuge for 20 minutes at 2,000xg.

Isolation of Cyclospora from Fresh Produce Washes

1. Aspirate supernatants (without disturbing debris pellets) to a volume not to exceed 45 ml when combined.
2. Suspend pellets in remaining supernatants and combine.
3. Add 2.5 ml 20% (w/v) celite in NET-BSA suspension (ensure that celite is thoroughly suspended prior to its addition to sample washes). Mix samples on a rotating wheel at room temperature for 15 minutes.
4. Add 1.0 ml of 10% PVPP suspension (w/v). Mix samples on a rotating wheel at room temperature for 15 minutes.
5. Prepare a 150 ml analytical filter unit by removing the membrane filter (0.45 µm or 0.2 µm) but retaining the grade 4 filter backing. Attach to a vacuum source.
6. Pre-wet the analytical filter with a small volume (~10ml) of NET buffer.
7. Decant celite/PVPP-containing sample wash into a prepared 150 ml analytical filter unit and vacuum filter. Ensure that the liquid passes through the filter septum to remove particulates and the celite particles from the suspension as it is filtered. The adsorbant celite should prevent filter clogging.
8. Rinse the container with 10 ml NET to recover as much of the residual celite-containing sample and decant into the filter unit. Then rinse the celite and particulate material trap onto the filter with an additional 10 ml volume of NET.
9. Prior to FTA filtration in step 4l, save ~10% of the filtered sample for microscopic examination.
10. Prepare filter funnel unit(s) containing FTA filter disk and attach to vacuum manifold.
11. Under vacuum, pre-wet the FTA filter assembly.
12. Slowly decant filtrate from step 4i into filter funnel unit while under vacuum until entire sample has passed through FTA filter.
13. In succession while filter funnel unit is still attached to vacuum manifold, rinse filter twice with 10 ml of FTA purification buffer and twice with 10 ml of FTA filter wash buffer.
14. Remove filter funnel unit from vacuum manifold, disassemble unit and dry FTA filter disk on 56°C heating block.

Isolation of Cryptosporidium spp. from Fresh Produce Washes

1. Aspirate supernatants (without disturbing debris pellets) to a volume not to exceed 10 ml when combined.
2. Suspend pellets in remaining supernatants and combine. NOTE: pellet volume should not exceed 0.5 ml packed volume as it will interfere with subsequent steps that employ immunomagnetic adsorption of Cryptosporidium oocysts.
3. Follow directions accompanying Dynabead anti-Cryptosporidium Kit for immunomagnetic separation (IMS) of Cryptosporidium oocysts from fresh produce washes using recommend tubes and magnetic capturing devices.
4. Elute captured Cryptosporidium oocysts in a 0.1 ml volume of 0.1N HCl for 5 minutes a room temperature.
5. Neutralize acid eluates with 0.01 ml 1N NaOH. Save ~10% of the sample for microscopic examination.
6. Dilute with 10 ml NET buffer.
7. Proceed as in Section 4A, steps j-n.

5 . Isolation of Parasitic Contaminants from Juices, Cider and Milk

This procedure can be used to analyze for contaminants in liquid samples such as orange juice, apple juice, apple cider, milk and milk products.

Isolation of Cyclospora from Juices, Cider and Milk

1. Adjust a 25 ml aliquot of liquid sample to pH 8.0.
2. Add an equal volume of NET buffer and mix well.
3. Proceed as in Section 4A, steps c-n

Isolation of Cryptosporidium spp. from Juices, Cider and Milk

A 10 ml volume of cider, juice or milk product is directly sampled by IMS using the Dynabead anti-Cryptosporidium Kit along with the recommend tubes and magnetic capturing devices as in Section 4B, steps c-f.

6. Isolation of Parasitic Contaminants from Large Volumes of Water

This procedure is designed to isolate contaminants from a designated water source (stream, river, reservoir, standing water, runoff, etc).

1. Place a single Envirochek™ sampling capsule in line with the water source to be sampled. Ensure that the flow rate does not exceed the standards established by the manufacturer.
2. Collect a water sample from 10 L of flow through.
3. Remove contaminants captured by the filter using 125 ml of elution buffer. Filters should be treated with elution buffer, sealed and agitated on a rotating wheel (moderate speed) for at least 5-10 minutes following the manufacturer's recommendations. Decant filter rinse into a 250 ml conical centrifuge tube.
4. Repeat step 6c and combine filter rinses.
5. Centrifuge the sample at 1,500-2,000 x g for 20 minutes allowing the centrifuge to coast to a stop (do not use the brake)
6. Proceed as described in Sections 4A and 4B for the isolation of Cyclospora and Cryptosporidium spp. respectively.

7. Slide Preparation and Microscopic Analysis – Cyclospora cayetanensis

Cyclospora oocysts emit a cobalt blue autofluorescence with the UV-1A emission filter or blue-green with broader emission spectra filters under ultraviolet illumination. Prepare slides in duplicate and examine slides under ultraviolet illumination as described below.

Laboratories should use a microscope reticle capable of measuring organism in the 8-10 µm range to verify oocyst size when organisms are recovered. Compare morphological characteristics of presumptive oocysts to those in a known standard.

Slide Preparation

Centrifuge the volume set aside for microscopic analysis (Section 4A, step i), at 1,500 × g for 10-15 min at 4°C. Aspirate supernatant to within 0.5 ml of debris pellet. Uniformly suspend pellet material by gentle, repeated pipetting.

1. Apply silicone vacuum grease to edge of cover slip
2. Place 10 µl of suspended debris to a clean glass microscope slide and prepare a wet mount using pre-greased cover slip 

Microscopy

1. Examine slide under UV light at 400× magnification. Cyclospora oocysts autofluoresce cobalt blue. Examine slide at multiple planes under UV. Occasionally, debris in slide preparations make it difficult to only view the slide on one plane.
2. Switch from epifluorescence microscopy to bright field microscopy or differential interference contrast microscopy. Determine oocyst size of any presumptive oocysts at 1000X magnification. Compare to standards. Confirm internal structures of presumptive Cyclospora oocysts as compared to a standard.
3. Seal cover slips to the glass slides of presumptive positives with fingernail polish, slide compound or paraffin wax.
4. Document presumed positive samples with photographs taken at multiple planes.

8. Slide Preparation and Microscopic Analysis & Cryptosporidium spp.

Microscopic examination of produce washings and other liquid samples for the presence of Cryptosporidium spp. oocysts will be conducted using commercially available immuno-magnetic bead separation (IMS) kits and immunofluorescence labeling kits.

1. An aliquot (~10µl) of the IMS-derived sample Section 4B, step e, is FITC-labeled using the Hydrofluor Combo Detection Kit for Cryptosporidium and Giardia (Ensys Inc., Research Triangle Park, NC) per the manufacturer's instructions and examined in conventional DIC and epifluorescence mode. 
    

   Table 1: Parameters for Epifluorescence Microscopy^†

   INCIDENT LIGHT
   Light Source-Mercury Vapor
   200W, 100W, or 50W

   Excitation Filter	Dichroic Filter	Barrier Filter	Red Suppression Filter
   KP500	TK510	K510 or K530	BG38
   FITC	TK510	K530	BG38
   D. Tungsten-Halogen 50 and 100 W
   KP500	TK510	K510 or K530	BG38
   FITC	TK510	K530	BG38

   ^†Taken from protocol provided with HYDROLFUOR-Combo Detection Kit for Giardia cysts and Cryptosporidium oocysts (Ensys Inc., Research Triangle Park, NC)

9. PCR Analysis

The molecular detection of Cyclospora spp. and Cryptosporidium spp. is independently accomplished using nested PCR protocols. The differential identification of Cyclospora cayetanensis from other closely related non-human pathogenic parasites (i.e. Eimeria spp.) employs a nested multiplex PCR assay. This assay can be accomplished using either a conventional thermal cycler with heated lid or a real-time PCR platform using the Roche LightCycler^®.

The detection of Cryptosporidium spp. also involves nested PCR amplification. However, differentiation and speciation of Cryptosporidium spp. requires further analysis by a restriction fragment length polymorphism (RFLP) assay. Please note the following: C. parvum genotype I has been renamed C. hominis; C. parvum genotype II (bovine strain) is now referred to as C. parvum

A. DNA Primers

^†All primer sequences were derived from the published sequences for the 18S rRNA genes of the respective organisms.

^†All primer sequences were derived from the published sequences for the 18S rRNA genes of the respective organisms.

B. General Sample Preparations for Primary PCR Amplifications

1. Punch marked triplicate areas (6 mm diameter) from dried FTA filter disk using a single punch hole-puncher. Decontamination of the hole-puncher is not necessary as cross contamination between samples from the hole-puncher has been found to be negligible. However, the individual researcher may wish to swab the punch with ethanol between sample disks if it is deemed appropriate.
2. Insert filter punches snuggly into bottom of 0.65 ml thin-walled PCR tubes.
3. Dispense 50 µl HotStartTaq™ Master Mix into each PCR tube.
4. Prepare reagent master mix (see Table 4) with the appropriate forward and reverse DNA primers (see Tables 2 and 3) and dispense into each PCR tube.
5. All PCR analyses must include positive and negative controls (see Table 5).
6. Mix tubes with gentle tapping.
7. Follow the appropriate thermal cycling protocol (Table 6 and 7) for primary PCR amplification.

   Table 4: General PCR Conditions for Primary PCR Amplification

   Component		Volume (µl)*	Final Concentration
   FTA Filter Disk (DNA Template)
   HotStartTaq™ Master Mix		50.0	†
   Reagent Master Mix
   	MgCl2, 25 mM	2.0	2.0 ‡
   	Forward Primer, 10 µM	2.0	0.2 µM
   	Reverse Primer, 10 µM	2.0	0.2 µM
   	Sterile deionized water	44.00

   ^*100 µl total volume
   ^†Final concentrations for components in the HotStartTaq™ Master Mix are as follows: 200 µM of each dNTP, 1.5 mM MgCl₂ and 2.5 U HotStarTaq™ DNA Polymerase
   ^‡Final MgCl₂ concentration is that contributed by both the HotStartTaq™ Master Mix and 25 mM MgCl₂stock

   Table 5: Controls for PCR amplifications

   Control Type		Condition/Organism
   Negative Control-1		Reagent blank-no filter
   Negative Control-2		Reagent blank + unspotted, washed filter
   †Positive Controls:
   	Cyclospora Analysis:
   		C. cayetanensis
   		‡¶Cyclospora spp. (NHP)
   		*Eimeria spp.
   	Cryptosporidium Analysis:
   		¶C. hominis (formerly know as C. parvum genotype 1)
   		C. parvum, (formerly know as C. parvum genotype II (Bovine)
   		¶C. baileyi
   		¶C. serpentis

   ^†Whenever possible, positive control FTA filters should be spotted with at least 10³ organisms
   ^‡Non-human primate-derived Cyclospora spp.
   ^¶Not routinely available.
   ^*Most available Eimeria spp. are suitable.

   Table 6: PCR Thermal Cycling Parameters for Cyclospora and Eimeria spp.

   	Step	Number of Cycles	Temperature and Time
   Primary PCR	Initial Activation	1	95°C; 15 min
   	Amplification	35	Denaturation: 94°C; 30 sec
   			Annealing: 53°C; 30 sec
   			Extension: 72°C; 90 sec
   	Final Extension	1	72°C; 10 min
   †Nested Multiplex PCR	Initial Activation	1	95°C; 15 min
   	Amplification	25	Denaturation: 94°C; 15 sec
   			Annealing: 66°C; 15 sec

   ^†This is a stringent, 2-step amplification program (simultaneous annealing and extension at 66°C). Likewise, it does not require a final extension step.

   Table 7: PCR Thermal Cycling Parameters for Cryptosporidium spp.

   	Step	Number of Cycles	Temperature and Time
   Primary PCR	Initial Activation	1
   	Amplification	40	Denaturation: 94°C; 45 sec
   			Annealing: 53°C; 75 sec
   			Extension: 72°C; 45 sec
   	Final Extension	1	72°C; 7 min
   Nested PCR	Initial Activation	1	95°C; 15 min
   	Amplification	35	Denaturation: 94°C; 25 sec
   			Annealing: 65°C; 25 sec
   			Extension: 72°C; 25 sec
   	Final Extension	1	72°C; 7 min

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