Behavior and Viability of Third-Stage Larvae of Terranova sp. (Type HA) and Anisakis simplex (Type I) Under Coolant Conditions

Abstract

This study reports effects of storage at cold temperatures on behavior and survival of third-stage larvae of Terranova sp. (type HA) and Anisakis simplex (type I) in marine fishes. Snappers, caught near the Hawaiian Islands, were examined to determine whether type HA and type I larvae could migrate from the viscera of ungutted fishes into edible musculature when maintained at 12, 8, and 0°C. Our data are suggestive that both type HA and type I larvae possess the ability to migrate. Temperatures of 12, 8, and 0°C had no noticable adverse affect on viability of both larval types within fish tissues; however, both larval types were extremely sensitive to temperatures below freezing. Death of both larval types encysted within Hawaiian snappers occurred by day 4 at-5°C and within 24 h at-10,-15, and-20°C. Other type I lar-vae, collected from fishes (Sebastes spp.) imported to Hawaii from the western Pacific, survived for slightly longer periods at-5,-10,-15, and-20°C when compared with type I larvae from Hawaiian fishes. Subjecting Hawaiian snappers to at least-20°C for 1 d and imported rockfishes to at least-20°C for 5 d is recommended to inactivate the living anisakines before ingesting any raw fish products. Anisakiasis is a disease induced by larval ascaridoid nematodes that have penetrated into or through the gas-trointestinal tract of m a m m a l s. H u m a n s m a y acquire this disease by c o n s u m i n g infected raw or inadequately cooked fishes or other seafood products. Most infective third-stage larvae, fortunately, reside in the viscera of fishes; not in the edible tissue. H o w e v e r , some studies (13,15) have demonstrated that under coolant conditions used to preserve freshly caught fishes, some anisakine larvae m a y migrate from the abdominal mesentary of intact fishes into the musculature. This migration substantially increased the risk for infection of consumers. Deardorff et al. (4) demonstrated that Terranova type A (H A) , third-stage larvae collected from the viscera of Hawaiian snappers, possessed the ability to invade the gastrointestinal tissues of experimental animal hosts. Because of the pathogenic potential of this parasite, the behavior and viability of these larvae at various coolant temperatures were investigated. Lut-janids were selected for these experiments because they are prized food fishes in H a w a i i , often eaten raw, and infected with both type H A larvae and the third-stage larvae of Anisakis simplex (type I) (5). The latter larval type has been demonstrated to migrate under similar experimental conditions. MATERIALS AND METHODS All local snappers were collected with the help of commercial and sports fishermen using hook and line during 3 February through 3 De-cember 1982. Because of the methods employed to catch these deep-reef fishes, the stomach of some snappers was inverted and protruded from the mouth-cavity. In these instances, the stomach was returned to its normal position in the body-cavity before experimentation. All fishes were stored on ice until used. Rockfishes (Sebastes spp.), caught by commercial fishermen in the western Pacific ocean, were immediately iced and shipped to us in Hawaii. For larval identification, some larvae were fixed in glacial acetic acid, stored in a solution of 5 parts of glycerin plus 95 parts of 70% ethyl alcohol, and later examined in lactic acid. To study the effects of freezing temperatures, whole fishes, both imported and local, were wrapped individually in plastic bags and placed in freezers previously set at 0,-5,-10,-15, and-20°C. If freezer temperatures , read twice daily, varied more than one degree, the experiment was terminated and repeated. Since the experimental design was to mimic standard home and commercial freezing procedures, temperature gradients within each fish were discounted. Fishes were removed from freezers at predetermined intervals, allowed to thaw at ambient temperatures, and digested as stated below. All recovered larvae were identified, counted, and assessed for viability. If a larva moved after being probed with a dissection needle, it was considered alive. Whole fishes were maintained at 12, 8, and 0°C for the migration experiments. Fishes were examined over a 21-d period. Once the "shelf life" of the fishes was exceeded at a specific temperature, experimentation was terminated. For each fish, the musculature (divided into sections) and viscera were digested separately. Tissues from the fishes were placed into a digest-solution contained within 500-or 1000-ml beakers, maintained at 35-38°C and gently agitated by magnetic stirring rods for 4-8 h. The digest-solution was 1% pepsin (w/v) with 0.7% HC1 and 0.85% NaCl. The solution was allowed to settle, decanted, and the remaining residual tissues examined for larvae. The 158 fresh snapper examined over a 26-month period (5) as well as periodic examination of 15 other snap-pers upon capture during this study, represent baseline data. In these fishes, no larvae were found in the musculature.