OPFLP-02 Isolation and Identification of Anisakid Roundworm Larvae in Fish
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E17C48DFDE6E40F68C78528D16721E00 |
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文件大小(MB)： |
0.06 |
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页数： |
7 |
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日期： |
2012-3-3 |
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Published on the Food Directorate’s (Health Canada's) website at:,http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html,Government of Canada Gouvernement du Canada,Laboratory Procedure OPFLP-2,June 2006,HEALTH PRODUCTS AND FOOD BRANCH,OTTAWA,ISOLATION AND IDENTIFICATION OF ANISAKID ROUNDWORM LARVAE IN FISH,Brent R. Dixon,Bureau of Microbial Hazards, Food Directorate,Postal Locator: 2204A2,Health Canada, Ottawa, ON, K1A 0L2,E-mail: Brent_Dixon@hc-sc.gc.ca,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,marine fishes. These procedures are meant for use by laboratory and inspection staff, and are intended to be,used for small quantities of fish originating from consumer complaints, as well as for larger samples collected in,routine inspections. This revised method replaces ExFLP-1, dated September 1995.,2. PRINCIPLE,The procedures involve manual, sedimentary, or chemical separation of roundworm larvae from fish tissues.,They are either non-destructive (candling and ultraviolet illumination), 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. Illumination 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 (10.2). 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,OPFLP-2,- 2 - June 2006,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.,3.2 Larvae of Public Health Importance,The larvae of two anisakid roundworms, Anisakis simplex (herring worm), and Pseudoterranova decipiens,(seal worm), have been implicated in the majority of cases of human anisakiasis. The infective larval stage,may be found on the viscera or in the flesh of a number of commercially important fish species, including,salmon, cod, herring, and mackerel, as well as squid (10.1). P. decipiens is also found in smelt and various,flatfish. Two other anisakid roundworms, Contracaecum osculatum and Phocascaris spp., have also been,identified in commercially important marine fishes and may be of public health concern.,4. MATERIALS AND SPECIAL EQUIPMENT,4.1 Candling,4.1.1 candling light table as described below,4.1.2 small forceps and culture dish containing normal saline (0.85 g NaCl per 100 mL distilled,water),4.2 Ultraviolet Light Illumination,4.2.1 hand-held or fixed ultraviolet light illuminator,4.2.2 face-shield or goggles (UV light resistant),4.2.3 small forceps and culture dish containing normal saline,4.3 Elution,4.3.1 U.S. Standard No. 4 sieve (or similar sized kitchen s……
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