고등어에서 분리한 고래회충 유충 : 형태학적 분류, 배양 및 항원성단백질의 분리 = Anisakid larva from mackerel : Morphologic classification, culture in vitro and antigenic characterization

다국어 초록 (Multilingual Abstract)

Eighty six cases of human anisakiasis have been reported since 1971 in Korea, but there might have been more cases of the Anisakis infection due to food habit of this country. This study was conducted to identify morphologic features, changes of larva after cultivation in vitro and antigenic profiles of Anisakid larva and excretory and secretory products. We purchased 27 mackerels from a market in Seoul, dissected the fish and collect the anisakid larvae. Microscopic examination of larvae was done after fixation(FAAG solution) and clearing(lactophenol solution). Total length and width, esophageal length(muscular and ventricular parts), character of head and tail of larvae were observed. Anisakid larvae in PBS solution were froze and thawed, 3∼4 times and the supernatant were collected after centrifugation at 3,000rpm for 30 minutes followed by another centrifugation at 20,000rpm for 1 hour. Larvae were cultured in MEM/5% FCS at 37℃, 5% CO₂incubator. Cultured medium were collected every day and concentrated through Centriplus(Amicon, M. W. ; 10,000) for excretory and secretory proteins. Both antigens were measured protein by Lowry method. Rabbirs were injected with larval antigen mixed with complete adjuvant intramuscularlly, 2 times for 3 weeks interval. Sera were collected before immunization, 3 weeks after 1st immunization, 2, 4, 6, 8, 10, 12 weeks after 2nd immunization. Serum IgG level were evaluated by ELISA method. Anisakid larva and excretory and secretory antigens were identified by immunoblotting.
All larvae from the mackerel were identified as Anisakis sp. type I. Anterior end of the worm showed a boring tooth on the lips. Esophagus was formed a muscular part and a ventricular part. Short tail was bluntly ended with a small sized and various shaped mucron. Nerve ring and excretory canal were noted anterior part of the worm. One week after cultivation, many worms were molted and lost boring tooth and tail mucron. Distinctly developed mouth part and rumpled intestine were noted. Cuticular striation was evident compared with that of the larvae before cultivation esp. esophageal region. Two weeks after cultivation, thickened cuticle, sigmoid ventricular part of esophagus, more rumpled intestine were obsered. Activity of the larvae almost stopped on this week. Serum IgG level began to elevate after 1st immunization and prolonged high titer to 12 weeks after 2nd immunization. Protein bands of 199kDa, 68kDa of crude somatic antigens and 100kDa, 77kDa of excretory and secretory(E/S) products reacted with immunized rabbit serum when 7.5% running gel was used on SDS-PAGE. Protein bands of 52 kDa, 20∼19kDa of crude somatic antigen and 50kDa, 45kDa, 23kDa, 23kDa, 20kDa of E/S product reacted with immunized serum on 12.5% running gel. Anisakis larva contained common antigens of Ascaris lumbricoides adult worm.