Chapter 19

Introduction

Humans unknowingly consume microscopic and small macroscopic animals with their food. The intestinal tract is inhospitable to most of these organisms, which are either digested or evacuated in the feces. However, some obligate or facultative parasites may become established in the human body. Although a number of parasites produce no symptoms and are not associated with disease, others may cause mild, moderate, or severely acute illness and even permanent damage. The following methods are used to examine foods and food-contact materials for the presence of parasites. For the most part, these techniques are labor-intensive and tedious; work continues to refine them and to develop additional techniques and rapid methods. Several alternative ways to examine fish and shellfish are presented. However, candling is the only method currently used for regulatory purposes with finfish.

Digestion to Select Mammalian Parasites in Edible Flesh

I.

By mimicking the chemical and temperature conditions of the mammalian stomach, this method frees parasites from the surrounding flesh and reduces the background of nonparasitic organisms.

Sampling and sample preparation

C.

From a sample weighing 1 kg, take a subsample (100 g) of beef, pork, or poultry, or 250 g of fish. Subsamples of most mammalian meat, poultry, or fish require no further preparation. They may be torn or separated into 5 or more pieces to increase the surface area. Samples with relatively large amounts of connective tissue are not readily digested; snail meat, for example, is digested very poorly. The following methods improve digestion. A 100 g sample is blended in 750 ml saline. Ten intermittent, instantaneous bursts in a blender will destroy some macroscopic organisms but usually will not affect microscopic organisms. A meat grinder is less destructive, although not suitable for some foods such as snails. Least destructive is initial digestion with papain followed by pepsin digestion.

Pathogens that are easily disseminated may be contained in samples and will be liberated by digestion. Of special concern are macroscopic tapeworm cysts and microscopic cysts of protozoa. When the presence of such pathogens is suspected, carry out the digestion and subsequent sample handling in a negative pressure hood until the suspect digest is placed in a safely closed dish. Handle all utensils as if contaminated, and autoclave or incinerate after use.

Digestion, sedimentation, and examination

D.

Adjust incubator-shaker or water bath to 37 ± 0.5°C. Prepare digestion fluid in 1500 ml beaker by dissolving 15 g pepsin in 750 ml saline, add sample, and adjust to pH 2 with concentrated HCl (about 3 ml). Place in incubator or water bath and stir (about 100 rpm) after equilibration for about 15 min; check and adjust pH again. Cover beaker with aluminum foil (if using stirrer, pierce hole for stirring rod) and continue incubating until digestion is complete. The time required for digestion will vary but should not exceed 24 h. Carefully pour beaker contents through sieve into tray. Rinse remains with 250 ml saline and add to digest. Examine rinsed contents of sieve and record results. Larger parasites will remain on sieve. Replace plug of sedimentation tube with rubber tubing and clamp folded tubing. Carefully transfer contents of tray to sedimentation cone. Transfer undigested sample or parasites to a petri dish, using spoon, tweezers, or dissecting needle. After 1 h of sedimentation, remove bottom 50 ml of sediment by releasing clamp and collecting in 100 ml beaker. Transfer fluid to petri dish(es) with eyedropper. (Digests vary in their clarity; if digest is dense, dilute with saline until it is translucent.) Cover dish and examine macroscopically for parasites; then examine with dissecting microscope and finally with inverted microscope (a contrasting background can be helpful). Count, tentatively identify, and record observations. Count total number of organisms and differentiate those that are living (motile) and dead (nonmotile), if possible. Examine complete contents of beaker. Light infections may require repeated sampling to detect parasites.

Interpretation and further identification

Further information about recovered organisms is usually required, both to classify them and to decide whether the criterion of movement is valid for determining viability. For example, the eggs of Ascaris must be "embryonated," i.e., allowed to incubate so that moving embryos develop inside. Cysts of some protozoa must be excysted to detect motion; those of Toxoplasma gondii can be judged viable only by the outcome of experimental inoculation into the peritoneal cavity of mice. Brief summaries of fixation and staining methods for frequently recovered parasites are given below and in the following references: invertebrates in general parasitology (2); animals' parasites (11); medical aspects of parasitology (10); food parasitology: methods, references, expert consultants (4,7); immunology and serology of parasitic diseases (8); protozoa (9); nematodes (3,16); trematodes (1,14); cestodes (15); arthropods (5).

Fixation and staining

E.

Protozoan cysts and helminth eggs. Fix and stain fresh material with Lugol's iodine solution (R40) or use fluorescent antibody stain (if available) on formalin-fixed material.

Nematodes

Fix in glacial acetic acid overnight and store in 70% ethanol with 10% glycerin. Study nematode morphology in temporary mounts by removing from alcohol and clearing in glycerin, lactophenol, or phenol ethanol. Before returning to storage, rinse away clearing fluid(s) with 70% ethanol. Sectioning and staining may be necessary for detailed identification of nematodes.

Trematodes and cestodes

Before fixation, relax both trematode and cestode flatworms in cold distilled water for 10 min. Fix flukes (trematodes) in hot (60°C) 10% formalin. Fix tapeworms (cestodes) by adding 10× volume of 70°C fixative to the relaxing fluid, or dip them in 70°C water repeatedly; then fix in a mixture of ethanol, glacial acetic acid, and formalin (85:10:5) overnight. Store in 70% ethanol. Flatworms are usually stained and mounted as permanent slides, but some may require sectioning and staining for detailed identification.

Acanthocephala

Place in water to evert the proboscis. (Some proboscises may evert almost immediately; others require several hours. Do not extend over 8 h.) Fix in steaming 70% ethanol with a few drops of glacial acetic acid. Store in fixative or 70% ethanol. Acanthocephala may be stained and mounted as permanent slides or, like the nematodes, cleared in phenol or glycerol.

Arthropoda

Fix fleas, lice, mites, copepods, fly larvae, and other parasitic or food inhabiting arthropods in hot water. Store in 70% ethanol.

Viability determination

F.

The major criterion for the viability of helminths is spontaneous movement. Observe organisms for 10 min to see if spontaneous movement occurs. If autonomous movement is not observed, touch with a dissecting needle and observe to see if movement has been stimulated. Allow specimens from salted products to equilibrate in at least 20× volumes of physiological saline for 3 h before viability determination; osmotic pressure may cause apparent movement. Culturable protozoa should be cultured in vitro to determine viability. If culture is not feasible, dye exclusion is the method of choice for viability determination.

Candling to Detect Parasites in Finfish

II.

See Attached

Compression Candling: Detection of Parasites in Molluscs and other Translucent Foods

III.

See Attached

Mechanical Disruption and Sedimentation for Detection of Larval Parasites in Fish Flesh

IV.

See Attached

Concentration of Helminths and Protozoa from Vegetables

V.

Vegetables may become contaminated with parasitic organisms through contact with animal or human fecal material or through application of sewage-derived fertilizer to croplands (13). The method outlined below can be used to examine fresh vegetables for parasites. A similar method recovered Cryptosporidium sp. from 1% of water samples examined. Recovery from vegetables would be estimated at 1% or less. (A sample consists of five 1-kg subsamples.)

Equipment and materials

A.

1. Balance
2. Polypropylene beakers, 1 L
3. Sonic bath, about 2 L capacity
4. Centrifuge, large capacity, low speed with swinging bucket
5. Polypropylene centrifuge tubes, 50 ml
6. Eye dropper, polypropylene
7. Culture dish with 2 mm grid
8. Microscope slides

Reagents

B.

1. Lugol's iodine (R40)
2. Sheather's fluid (500 g sucrose, 320 ml deionized water, 6.5 g phenol)
3. Detergent solutions Nos. 1, 2, and 3 No. 1 - 2.5% formaldehyde, 0.1% sodium dodecyl sulfate (SDS), 0.1% Tween 80 No. 2 - 1% Tween 80, 1% SDS No. 3 - 1% Tween 80
4. Fluorescent antibody kit

Procedure

C.

Store vegetables in refrigerator before analysis. Separate vegetables into units: tight head type (cabbage), remove outer 3 layers of leaves; loose head type (leaf lettuce), separate individual leaves; root type (carrot), no preparation; floret type (cauliflower), separate into florets of about 50 g. *Pour 1-1.5 liters of detergent solution No. 1 into sonic bath and add vegetables loosely to about 250 g; operate bath for 10 min. Remove vegetables individually and drain well. Repeat from *, above, until subsample is completely sonicated.

Transfer detergent to beaker; then dispense all of the material into 50 ml centrifuge tubes. Centrifuge at 1200 × g for 10 min. Remove supernatant to 1.5-2 ml and consolidate sediment into one tube with eyedropper or plastic pipet. Rinse each tube twice with 1.5 ml detergent No. 2, and add to consolidation tube. Rinse and centrifuge sediment twice with detergent No. 2. Dilute to 10 ml with detergent No. 3 and sonicate for 10 min. Add 25 ml Sheather's fluid to clean centrifuge tube and layer on detergent suspension from sonic bath. Centrifuge at 1200 × g for 30 min. Remove 7 ml of fluid from interface and transfer to centrifuge tube; fill tube with detergent; then centrifuge at 1200 × g for 10 min. *Remove supernatant and dilute with detergent No. 3. Then centrifuge for 10 min at 1200 × g. Repeat from *, above, 2 times.

For helminth eggs, transfer sediment to gridded petri plate and add 1 ml Lugol's iodine. Dilute and examine entire plate with inverted microscope. For protozoa, dilute sediment sufficiently with detergent No. 3 to make translucent 100 µl thin smears on cleaned polylysine-coated microscope slide cleaned with acid alcohol.

Let slides air-dry. Add positive and negative control samples to separate well or slide and let air-dry. Follow manufacturer's instructions for fluorescent antibody staining. Examine each slide at 200-300× with fluorescent microscope. Record results. If sample is positive, calculate number of cysts present per kg of food specimen by measuring remaining suspension and estimating number. If sample is negative, stain and examine remaining sediment.