Isolation and Identification of Anisakid Roundworm Larvae in Fish

Laboratory Procedure exflp01
September 1995

1. Application

These laboratory procedures are applicable to the isolation and identification of live third-stage anisakid roundworm parasites commonly found in the flesh or on the viscera of a variety of commercially important salt water fish. These procedures are intended for use with small quantities of fish originating from consumer complaints, as well as for larger samples collected in routine inspections. This revised method replaced exflp01, dated September 1994.

2. Principle

The procedures involve manual, sedimentary, or chemical separation of roundworm larvae from fish tissues. They are either non-destructive (candling and ultraviolet irradiation), or destructive (elution and digestion) techniques. Identification is based on gross morphological features which may be observed microscopically in live or preserved specimens. Candling is currently the only procedure used in routine inspections at federally regulated processing plants. Irradiation with ultraviolet light is another laboratory method used for detecting anisakid larvae at, or near, the surface of the flesh or viscera by their fluorescence. Although it will kill any live parasites, detection through exposure to ultraviolet light is improved by a preliminary freeze/thawing of the specimen.

Elution involves the migration of some (but not all) live parasites into saline while, in digestion, all parasites, live or dead, are isolated. While no significant difference in the total number of worms recovered by either technique was observed, the numbers of pathogenic worms (eg. Anisakis and Pseudoterranova) recovered by the digestion method can be 175% higher than the number recovered by elution (10.6). As the parasite yield may be improved somewhat, a combination of candling, elution, and digestion is recommended.

3. General Characteristics of Anisakid Roundworms

3.1 Life Cycle

The anisakid roundworms include a number of closely related genera within the phylum Nematoda, with a relatively complex life cycle (Fig. 1) involving a free-living stage and multiple hosts. Eggs released from the mature worms are passed in the faeces of marine mammals, which act as the definitive hosts. The eggs sink to the sea floor and hatch into second-stage larvae within days or weeks depending upon the water temperature. These larvae then rely upon ingestion by marine crustaceans in order to facilitate their continued development into the third-stage. When the crustacean is eaten by a fish or squid the larvae migrate into the tissues of this second intermediate host and develop to the advanced third-stage on the viscera or in the muscle. When an infected fish is eaten by a definitive host such as a marine mammal, the larvae are released into the stomach or intestine where they undergo further moults, developing into fourth-stage larvae and eventually adults. Humans can only be considered accidental hosts in this life cycle, and have no influence on the transmission of these parasites.