AR CHrVE S FISHERIES AND MARINE SERVICE. Translation Series No Anisakiasis as a parasitic zoonosis and its prevention. 2. by Y.

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1 AR CHrVE S FISHERIES AND MARINE SERVICE Translation Series No Anisakiasis as a parasitic zoonosis and its prevention. 2. by Y. Ono Original title: Jinchiku kyotsu kiseichubyo Anisakis-sho to sono taisaku - 2 From: Chikusan No Kenkyu 29(5): , 1975 Translated by the Translation Bureau grm) Multilingual Services Division Department of the Secretary of State of Canada Department of the Environment Fisheries and Marine Service Pacific Biological Station Nanaimo, B.C pages typescript

2 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION CANADA SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE Japanese AUTHOR - AUTEUR 0110 Yutea TITLE IN ENGLISH - TITRE ANGLAIS INTO EN English Anisakiasis as a parasitic zoonosis and its prevention - l'art 2 TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTLRES ROMAINS) jinehihu kyotsu kiseiehubyo Anisakis-sho to sono taisaku 2. REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGbE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACT'ERES ROMAINS. Chikusan no Kenkyu REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS Animal Husbandry PUBLISHER ÉDITEUR Yokendo lubl. Co., Ltd. PLACE OF PUBLICATION LIEU DE PUBLICATION Tokyo, JA1AN YEAR ANNÉE DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO ' PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL pp NUMBER OF TYPED PAGES HOMBRE DE PAGES DACTYLOGRAPHIÉES 21 REQUESTING DEPARTMENT MINISTÈRE-CLIENT Environment TRANSLATION BUREAU NO. NOTRE DOSSIER N 1101f30 BRANCH OR DIVISION DIRECTION OU DIVISION rialleripa & Marine TRANSLATOR (INITIA LS) TRADUCTEUR (INITIALES) KFM PERSON REQUESTING DEMANDÉ PAR YOUR NUMBER VOTRE DOSSIER N DATE OF REQUEST DATE DE LA DEMANDE Allan T. Reid 19 Octoher, 197t NOV UNEDITED TRAN.SLATIC.:N For inforrila:ion only TRADUCTION NON REVISEE Information sz,.ulorlent SOS 200.I 0..8 (REV. 2/66)

3 Rrc 5-9ÇT (6/76) r e, Secretary Secrétariat e'er of State d'etat ij TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES CLIENTS NO. DEPARTMENT DiviSION/BRANCH CITY NO DU CLIENT MINISTiRE DIVISION/DIRECTION VILLE Environment Fisheries & Marine Ottawa, Ont. eureau NO. LANGUAGE TRANSLATOR (INI rials) NO DU BUREAU LANGUE TRADUCTEUR (INITIALES) Japanese K.F.M. NOV Chikusan no Kenkyu (Animal Husbandry), Volume 29 (No. 5): pp , ANISAKIASIS AS A PARASITIC ZOONOSIS AND ITS PREVENTION - PART 2 Yutaka ONO* 2. Epidemiology of Anisakiasis Although gastrointestinal diseases in man due to larvae of the 'anisakis nematode appear to have existed all over the world for a long time, it was VAN THIEL et al. of Holland 'Who in 1960 discovered highly acidophiiic phlegmonous foci and larvae of this nematode in the stomach of 11 patients who underwent laparotomy due to acute abdominal symptoms. Similar larvae had been found in their laparotomized patients since 1955 (their first case), but the larvae had not been identified at all until However, because of a fondness of these patients for eating uncooked herring and because of the coincidence of the occurrence of these cases with the fishing season of herring; i.e., between May and August, it had been suspected that some parasite was responsible for the disease * Professor Emeritus, Kobe University (At present, Kobe Women's College) UNEDITED TRANSLATION For informwion only TRADUCTION NON REVISEE Information seuiemorit

4 a 2 and that the parasite originatesin herring. VAN THIEL at first identi- fied these larvae as Eustoma rotundatum which is known to parasitize herring. Upon further examination, he revised the initial nomenclature to conclude that the parasites were larvae of an Anisakis species (1962) and named the disease caused by these larvae 'anisakiasis'. Later ASHBY (1964) presented a treatise discussing clinical and pathological aspects of anisakiasis in 89 cases that were compiled from existing literature. His treatise drew a great deal of attention among the people of northern European countries including Holland. In Japan NISHIMURA (1963) identified worms obtained from small abscesses in the mesentery of a patient as Anisakis larvae, so did ASAMI (1964) with larvae found in a piece of the stomach cut off from a man. Similar cases of larvae infestation in patients were also presented by YOSHIMURA and YOKOGAWA (1964) and OZURU et al. (1964). In 1965 a comprehensive research project under the title of 'Studies on parasitic granuloma' (coordinated by Prof. M. OZURU) was initiated with the financial support of the Ministry of Education and since then active research in various fields including parasitology, pathology and zoology has been carried out. Also, a large number of papers have been published on this disease and in a few recent meetings of the Parasitology Society of Japan anisakiasis along with toxoplasmosis are the most attentionattracting topics. Of particular interest is the fact that both are zoonoses. 1) Occurrence Occasionally people experience gastrospasm or food poisoning-like symptoms after eating very fresh or almost fresh raw marine fish and such

5 symptoms have been considered to be not uncommon. During the recent decade, however, due to rapid progress in endoscopie and/or radiographic diagnoses, these symptoms are in increasing numbers being diagnosed as anisakiasis. For example:, it is possible to detect by endoscopy invasion of anisakis larvae into the stomach wall in a patient, then to take out these larvae with stomach biopsy forceps and identify the sampled larvae while they are still alive. Case histories involving such operation have been reported by clinicians in various districts of Hokkaido where anisakiasis occurs in the highest frequency. Nematodes in the genus of Anisakis are parasites living in such mammals as a variety of whale and dolphin species as their final hosts. However, since these animals are ocean inhabiting, information on parasitic conditions of,and changes due to the anisakis nematode in, these final hosts is scanty and only a few reports exist; i.e., OKAMURA et al. (1958), NISHIMURA et al. (1961), NAKAGAWA et al. (1965), KAGEI et al. (1966) and KIKUCHI et al. (1967). Of these, KAGEI and his group examined various species of whales and dolphins and found anisakis infestation in the rumen of 213 (73.4%) of a total of 290 striped dolphins. On the other hand, KIKUCHI et al. autopsied 72 dolphins that had been captured off the Izu Peninsula and then died while being reared in the Marine Land at Enoshima, found infestation in 16 dolphins and concluded that five of the 16 individuals had died of anisakis parasitization. In some cases large numbers from 100 to 200, even 300 in one case, of worms were seen being attached to the mucosa of the first and second stomach chambers, and individual worms were invading the mucosal proper membrane or the submucosal tissue.

6 at the later stage. 4 The mucosa at the site of attachment was swollen and hemorrhage, necrotization, erosion and ulceration were seen in many areas. The ulcers often were as large as the tip of a little finger. Histological changes in the first stomach chamber where the largest number of parasites were seen, were mainly characterized by neutrophilic and histocytic responses, but the formation of phlegmon and phlegmonous tumor accompanied by marked acidophilic infiltration which is a characteristic picture of anisakiasis in man was not seen. According to these authors, of interest was the tinding that inflammation in the second stomach chamber where anisakis infestation was less extensive',was similar to that observed in man. (a) Etiology of anisakiasis in man and allergy A number of investigators have proposed that manifestation of anisakiasis symptoms is associated with allergy. Such a relationship was pointed out by the first disoaverer of anisakiasis, VAN THIEL, already in 1960; i.e., he explained that local allergic reactions at the digestive tract caused by repeated ingestion of Anisakis larvae would induce anisakiasis. KOYANAGI (1967) carried out an experiment in which pups and rabbits were sensitized by intraperitoneal injection of Anisakis worms. When the sensiti.zed and nonsensitized (control) animals were allowed to be infested with Anisakis larvae, resulting pathological changes were in the two forms; those which appeared to have resulted from allergy and those which were characterized by the extension of granulation tissue-surrounded foci. He stated that allergy due to repeated ingestion of Anisakis larvae played a main role in the developmént of anisakiasis and that factors responsible for these pathological changes were related to secretions and excretions of the pnrasites at an early stage of the disease and to degenerated and disintegrated parasites

7 SUZUKI et al. (1970), based on their own as well as hitherto reported findings, have divided anisakiasis into two types. One type is (p.606) characterized clinically by such suddan symptoms as to require some operation of the digestive tract and histologically by phlegmon. These changes are conaidered to result from allergic reactions of the patients who have already been sensitized with Anisakis larvae toward reinvasion of larvae of the same species. The other type is seen in nonsensitized subjects in which larvae invading the digestive tract initially act as foreign matter and then begin to be engulfed by the surrounding connective tissue to disintegrate eventually. During this process the larvae induce local allergy reactions,resulting in complex histological pictures. (b) Patients distribution and clinical characteristics of anisakiasis ISHIKURA (1968) compiled clinical data up to 1967 on a total of 278 anisakiasis patients from major medical and research institutions across Japan as well as from his own clinics in Hokkaido and divided the cases into two groups, based on clinical symptoms related to the stomach or intestine, both of which are the main target organs of anisakiasis. The major symptoms of stomach anisakiasis are pains and feeling of inflation at the precordia, nausea, vomition, stomachache, and bumps at the precordia. However, 11.6% of the cases lacked any subjective symptoms. In intestinal anisakiasis on the other hand, pains in the lower abdomen, nausea, vomition and others resulting from irritation of the peritoneum were the common symptoms. In most cases the onset of these symptoms was sudden and very few had no subjective symptoms. He further divided stomach anisakiasis according to severity of the symptoms into two forms - severe and mild and also treated intestinal anisakiasis in the same

8 6 manner. It was found that considerably large numbers of cases that had originally been diagnosed as gastrosp3sn or food poisoning were actually classified in the severe form of stomach anisakiasis. Furthermore, ISHIKURA and TOTSUKA (1974) compiled cases that were diagnosed as anisakiasis at all medical colleges and major hospitals across the country during a five year period between 1968 and 1973 (Table 3). All of these cases had been confirmed either by recognition Table 3 Number of anisakiasis patients recorded in various districts of Japan (as of January 1973) 3v.L7 x \i^, -r a J.. t :T1; I ^ R ^t3 I ^ I t' A ar ra M 127(14) ^'r ^r CM 3k D $ M k àcc C& F * M C-r 'N M H jl, fn e`af7fe J it I363 I I 496 I II III IV Stomach anisakiasis Intestinal anisakiasis Other types of anisakiasis Sum A Hokkaido district B Tohoku district C Kanto district D Chubu district E Kinki district F Chugoku district G Shikoku district H Kyushu district I District unknown of larvae by X-ray endoscopy or surgical operation, or by patho-histological or parasitological diagnosis. According to this table, the cases of anisakiasis hitherto reported in Japan amounted to 496 (including the aforementioned 278 cases that were described by ISHIKURA). Of which, 363 cases (73.2%) were in the class of stomach anisakiasis, whereas 124 (25.0%) intestinal anisakiasis. In addition, there were other cases of anisakiasis including the caul or the mesentery, two for each, and those affecting the liver, pancreas, tonsillar fossa and unidentified site(s), one for each. When the numbers of anisakiasis patients in different districts of the country are compared, in every district there were more stomach anisakiasis than intestinal anisakiasis patients. Hokkaido led in the numbers of both types of anisakiasis, followed in

9 7 decreasing order by Chubu, Kinki and Tohoku districts. The high frequency of anisakiasis in the district of Hokkaido was presumably due to high infestation rate of Anisakis larvae among fish and shellfish in this district and also largely to the effort of local clinicians who were interested in this disease. This was reflected in particularly large numbers of anisakiasis patients reported from a few special hospitals in the district; 46 cases from the Hospital of the University of Hokkaido, 49 from Karasawa Hospital in Asahikawa City and 16 from Doi Hospital in Mihoro City. The frequency of anisakiasis peaked in May, but was-low in August and maintained mid values between October and March. Anisakiasis occurred every year of the five-year period. The frequency in both sexes was as follows: of a total of 460 cases, 317 were male and 143 female, the ratio being 2.2 : 1, and in every district there were more male than female patients. When the patients were classified according to occupation, every occupation was included and no particularly high frequency was observed in those who were in marine fishery or restaurant business. When the nationwide distribution of anisakiasis patients was examined, coastal areas showed sbmewhat higher than inland areas. However, high numbers were observed in some parts of a few inland areas. When the late survey was compared with the earlier one (ISHIKURA, 1968), it was apparent that some changes were taking place in the pattern of occurrence. Also, ISHIKURA (1968, 1974) touched upon the general host-parasite relationship by expressing the view that this relationship is fluid and is now in the process of changing and accordingly, people cannot be

10 Table 4 Sea food eaten by anisakiasis patients (January 1968 to January 1973) regarded as an exceptional host. In other words, it is irrational ----,f I,-ig to consider that people are the ) * t / / 32 t) 4 et n 5 I 5 9'e e u I 1 b) )77 4 t e ) À 5 roe v 4 2. z. c I C 1 3) 4 7 ::" I eetow î r..b mm )er À e, I Dp7ee,t 1 A Hokkaido B Tohoku C Kanto D Chubu E Kinki F Chugoku G Shikoku H Kyushu I Raw fish slices 1) Pacific halibut 2) Squid 3) Tuna 4) Flatfish Paralichthys olivaceus 5) Rock trout 6) Pacific cod 7) Plaice 8) Ocean perch 9) Atka mackerel 10) Spanish mackerel 11) Gray mullet 12) 'FU' jellyfish 13) Sardine 14) Source unknown (both lean and whitemeat) 11 Processed raw fish 15) Sliced and vinegared mackerel 16) Cod roe 17) Vinegared and salted herring 18) Vinegared and salted Atka mackerel 19) Vinegared and salted cod 2 _ 2 sole predetermined host of a given species of parasite and hence parasites of other species of animals can evolutionarily change original hosts. Accordingly, the existing relationship between larvae of the Anisakis genus and people may in future be altered and the same can be said for all classes of parasites. When this occurs hbwever, it is difficult to determine whether a parasite (p.607) species itself has changed its characteristics to infest several new hosts or whether the existence of these new hosts has been overlooked up until the time of observation. Of extreme interest from the evoluaticnary viewpoint is the possibility that a parasite may, by changing its characteristics, infest an animal species which has so far escaped

11 9 infestation by that parasite,and further long-term investigation will be required to validate this possibility. 3. Anisakis Infestation in Fish in Japanese Waters and Their Ovaries (Herring and Cod Roe) 1) Infestation in fish It is extremely important from a viewpoint of anisakiasis prevention to know exactly the species of fish that nematodes of the genera of Anisakis and Terranova infest. Such surveys have been carried out on various fish species extending over a considerably wide range. KAGEI (1970), based on a number of other investigators' reports, has prepared a list of fish species in Japanese waters that harbor the anisakis nematode. According to this lis4 type I Anisakis larvae are found in 132 fish and 1 squid species and type II AnisakiS larvae in 25 fish and 2 squid species. The list also contains numerous fish species for larvae of the genera of Terranova and Contracaecum. Type I Anisakis larvae are found largely among fishes which are distributed mainly in northern Japanese waters and in a group of fishes seasonally migrating north. For example, off Hokkaido sagittated. calamary grow into adult individuals in fall. Between October and November schools of adult calamary migrate south being divided in the Japan Sea and the Pacific sides to reach the southwestern Pacific where they spawn in February and March. Newly developed as well as some old individuals resume northward migration in May to June. Although anisakis infestation is rarely seen among those north-bound individuals which were born in the southwestern sea, those which are Caught off Hokkaido in fall and winter show high rates of anisais infestation. These

12 10 clearly indicate that the source of infestation lies in northern waters. The eating habit of fish and squid also markedly affects the extent of anisakis infestation. As shown in Table 5, of fishes in group A, Table 5 o Infestation of fish and squid with Anisakis larvae r.0. xit Pe- ' I..-1:41-M. t.. IA V- _.`"7-e"- =--U e â nmilef± m m Rem ±11 me,, /; L-coetto. lee9,x W 100 HA e no, ( 1 101**e X,1e4q., L t*,9e % % 4.5 * Ë 33 -Y f 9 e m LO '' ', c.). y aZ. Aal (B) 13*i01e (*Tre, -q. 7..) Eue 0 )K.5)eL. t / J'' i), 4rseeltE, -Fixe,44* * n -i"' to -1:- y,..e M 5 LO 0 0 ittle7'/yel.1, inlelt17'/ Ye r v, (c) eleil(r_e<oit l )7' e i 100 0? L? L. giznom-tt I, e, e e * tt to 1_ ? )et * tt (D) EI*;ditreittZte55Z I _ 4e",tr'Y'e'r J frxt*e enoto 1..)-r.=ev4 10 o o o o zl:tit lilt 4 * ? I? 1 f = *4* ? ? I Distribution (A) Mainly northern Japanese waters species (B) Coasts of Japan (The Japan Sea and the Pacific) and seasonal north-south migrating species (C) Throughout warm waters, but migrating large species (D) Japanese waters and relatively sedentary species II Species 1) pollack 2) cherry trout 3) Pacific cod 4) herring 5) horse mackerel 6) flat mackerel 7) sagittated calamary 8) mackerel pike 9) sardine 10) red sunfish 11) bonito 12) spottedmackerel 13) Sarda orientalis 14) tuna 15) Kareus bicoloratus 16) Limanda vokohamae 17) 'HI' squid 18) SONGO I squid 19) Sepia esculenta 20) Dorvteuthis bleekeri III Number of individuals examined IV Rate of infestation V Average number of larvae detected VI Intramuscular infestation VII Eating habit a) polyphagous b) crustaceans c) fish d) fish-eating e) zooplankton phytoplankton g) sessile food Ii) bottom-dwelling organisms * Mostly type II Anisakis larvae (KOBAYASHI & KAGEI, 1966 and later publications)

13 11 such polyphagous species as pollack, cherry trout and cod have high values for both the rate and the number of infestation, but herring that feed mainly on crustaceans show low values for both. The saine holds true for fishes in group B; i.e., while no infestation is seen in solely phytoplankton-feeding sardine, infestation, though very law, is observed in mackerel pike that are totally dependent on zooplankton. When individual fish of different ages are compared, in every species both the rate and the number of infestation increase with advancing age. In zooplankton-feeding mackerel pike infestation occurs late in their life - after two years old. On the other hand, infestation is established in horse mackerel immediately after two years of age when their eating habit changes from plankton to fish. Also, a very high degree of infestation is seen even among young individuals of cod and sea trout, both of which are polyphagous. In the fish body, Anisakis larvae are mainly located below the surface membrane of various organs inside the peritonal cavity, but the main site is the surface of the liver in pollack. A small part of these larvae also invade the interior of the muscle, creating a great danger as a source of infestation to man. Within the muscle tissue the larvae, when taking an encapsulated form, are spiral and only 3 mm in diameter. Hence they are easily swallowed without being detected as a host fish is eaten uncooked. According to a survey on the parasite location in fillets of arrowtooth flounder and _ltitstpk_ Reinharciiolossoides from the Bering Sea, the largest numbers of parasites were found in the area that were in contact with the viscera of the fish in 90% of the infested fillet samples and the number markedly decreased as the examination site was shifted from the abdomen to the

14 12 dorsal and the tail parts. This appears to reflect the sequence of invasion of the larvae; i.e., they enter the digestive tract of the fish with the prey and then move into the muscle through the abdominal cavity. Anisakis infestation in fish shows some seasonal fluctuations; the number of infestation is high in winter in horse mackerel, whereas the infestation rate is high in spring and summer in mackerel,but in winter in sagittated calamary. Generally speaking, infestation with Anisakis larvae is high in those fish which inhabit the North Pacific, especially northern Japanese waters, and which migrate seasonally, but (p.638) tends to be low in warm water-inhabiting or sedentary fish. 2) Infestation in herring and cod roe As has been aforementioned, it was VAN THIEL (1960) who first discovered anisakis infestation in man. Based on the fact the the anisakiasis patients were fond of eating raw herring, he examined herring for parasites and finally confirmed the pathogen of anisakiasis. However, so far very few observations; only two - KHALIL (1968) and YAMADA (1971), have been carried out to ascertain whether roe, corresponding to the ovary, of herring is infested with Anisakis larvae or not. Recently KAGEI and IMAYE (1971) detected a large number of Anisakis larvae in herring roe samples that were placed on sale in Kyoto (Fig. 4). These samples originated from fish that had been captured in an ocean outside Japanese waters. The authors have warned about the danger of this new food sanitation problem. Of the 82 salted samples collected, 33 (40.2 7e) contained larvae and the average number of larvae infesting per roe sample was.2.8 (the maximum was 21). These larvae were located

15 13 in a greater number of the samples, 60 (69.8%) of the 85, on the side facing the intestine rather than close to the abdominal cavity of fish. Anisakis larvae were also seen in fairly large numbers at the head and tail parts of the ovary samples. Nearly all of the larvae found were located on the surface of the ovary forming a spiral, and only in 6 (7.3%) of the 85 samples they invaded the interior of the ovary. These findings suggest that in the ocean Anisakis larvae are transferred from their paratenic host (krill) into fish (herring), then pass through the intestinal wall. When they become encapsulated, this takes place in the largest numbers on the surface of the ovary, close to the intestine. The reports concerning infestation with Anisakis larvae of cod roe that as herring roe, is eaten uncooked after storage in a salted form are also very few. and there are only three. In one of these, HAYASAKA et al. (1969) described I \' " I' s'e ç;. 67." (- Fig. 4 Anisakis larvae isolated from salted herring roe samples a patient who developed gastralgia-like symptoms shortly after eating cod roe. Live Anisakis larvae were discovered in remnants of the cod roe that this patient had eaten and the case was clinically diagnosed as intestinal anisakiasis prior to operation. They have pointed out the dangerousness of such salted fish roe preparations. Fish ovarian food, like herring and cod roe, is usually processed as follows: parent fish (herring and various species of cod) are opened to separate and collect ovaries which are then bleached with hydrogen peroxide, treated with salt water, salted and finally frozen for

16 14 storage. Although most larvae in these salted ovaries are killed, as has been reported by HAYASAKA et al., some larvae occasionally survive and can parasitize man. It should be noted here that since anisakiasis is considered to be one of the allergic diseases, even dead larvae, once inside the human body, can sensitize the host (man) so as to cause severe allergic reactions upon reinfestation. Thus eating uncooked fish roe is a hazard (ISHIKURA et al., 1965; SUZUKI et al., 1970) and certainly poses a problem from a food sanitation viewpoint. 4. Food Sanitation Measures There is a possibility that nearly all of inshore fishes which are used for food in Japan are infested with Anisakis larvae. Furthermore, anisakiasis is reported not only in Japan but also in some Asian (Korea and Taiwan) and European countries including Holland, the U.K., Germany, France, Norway and Denmark. Hence establishing anisakiasis control measures should be regarded as an international project. Since anisakiasis has recently and suddenly moved into the scientific limelight and since it originates in marine fish and shellfish which have hitherto been regarded as being almost unrelated to the disease, its etiology is only poorly understood. Accordingly, food sanitary measures aiming at complete eradicationand/or prevention of Anisakis larvae have not been provided for at present. In Holland where anisakiasis was first discovered, a law concerning the prevention of anisakiasis was established already in However, since in that country the herring is the onlyspecies of fish that is eaten raw by people, enforcement of the law appeared to have been relatively easy, and it is said that since..the enactment of this law,

17 15 anisakiasis has almost been undetected. The circumstances in Japan, however, are far from easy and measures that are deemed feasible at present are as follows: 1) Infestation preventive measures for fish food (a) Keeping: from eatin.g- raw fish One should avoid eating raw sea fish and/or squid in any form, be it plain, cold water-washed or vinegared, and this is the first line of defence against infestation. However, fish and mollusks constitute one of the indispensable protein sources in the Japanese diet and also (p.609) they provide a means by which fishermen can obtain a livelihood. Of various fishes, the most important as the infestation sources are mackerel, horse mackerel, herring, pollack, tuna and sagittated calamary and hence one should be cautious of the danger of eating these fishes uncooked. Also, when fish are inspected in the close-to-fresh state, every effort should be made to remove parasitic larvae (the methods will be discussed later). This practice has been strictly carried out on fish meat materials by food processing factories handling fish blocks. However, when fish meat samples are more than several mm thick, larvae parasitizing such samples easily escape detection and complete elimination is difficult to accomplish. (b) Heat treatment Anisakis larvae are extremely vulnerable to high temperature and are killed after about an hour at 45 0 C but 2 min at 50 0 C(40% will survive for 1 min), several seconds at 55 to 60 0 C and instantly at over 65 o C. Therefore cooking fish with heat is the most effective way of

18 o 16 food sanitation against Anisakis larvae. (c) Low-temperature treatment (Ice packing and Freezing) In one of KAGEI's papers, the data of Gustafflin (1953) of Denmark on the survival rate of Anisakis larvae at cold temperatures were quoted as follows: 5.5 7e after 5 days at -5 C; and on the 5th and 10th days at -10 o C, respectively; and 07 after 2 min at -20 o C. These data should be interpreted with caution, since the conditions used in this observation were different from the actual condition of the larvae within the fish body. PICK of Holland also has stated that freezing fish for 24 hrs prior to use would eliminate the danger of parasitization without affecting the taste of the fish. However, when the fish were stored under an ice-packed condition, all larvae survived and were active (Table 6). Table 6 Survival rate of Anisakis larvae under different storage conditions (KAGEI) gifr2 Ytxxii. -re- >75 In Holland, freezing herring at -20 C for 24 hours is legally defined as the preventive measure against anisakiasis. However, OISHI has casted some doubt on the safety of this procedure by stating that a period of 24 hours may not be sufficient to I Storage conditions and fish species II No. of larvae dead III No. of larvae alive IV Packed in ice a) Horse mackerel b) Mackerel c) Sagittated calamary d) Total V Frozen kill completely Anisakis larvae and added one processor's observation in which larvae survived after exposure to C for 24 hr. Also, there is a report that larvae adhering to cod roe were still alive after storage at C for one month. Huwever, it remains to be answered whether this report is reliable.

19 17 At present, freezing is regarded as the most promising preventive measure against infestation with Anisakis larvae. Hence the finding of larval survival at C will markedly influence the outcome of any future preventive and/or sanitary measures and accordingly, it is desirable to make a scrutiny into this finding at tie earliest opportunity. (d) Ultrasonic treatment When larvae freely swimming in physiological saline solution are exposed to 9 kc ultrasonic waves, the larval body is destroyed within 2-3 min. However, this treatment is almost ineffective to largae sandwiched between fish meat (OISHI et al.). (e) Germicidic lamp treatment Radiation of larvae swimming in physiological saline solution with a commercial germicidic lamp for 24 hr results in marked loss in motility but no death of the larvae. The same treatment is totally ineffective against larvae when placed between fish meat (OISHI et al.). (0 Chopped larvae Larvae in the fish body may be cut while the fish is being cooked and it has been questioned whether these chopped larvae, when taken into the hgman body, are-able to establish parasitization. According to one theory (ASAMI, NISHIMURA), Anisakis larvae, even when cut in half or quarter, can maintain parasitizing ability.- This theory, however, has been totally denied by others (MATSUOKA et al., YAMAGUCHI). (g) Seasonings and food It is said that a solution of Japanese horse-radish (effective component is isothiocyanate), the subject of recent public conversation, in a concentration of 1/10,000 kills Anisakis larvae within 30 to 60 min

20 18 and that in a 2% aqueous solution of commercial horse-radish powder product they are killed after one day. It would, of course, be ideal, if application of this seasoning to sliced raw fish on the table killed instantly the larvae. However, the real value of Japanese horse-radish as a larvicide is questionable. Anisakis larvae survive for more than 175 days in 1-2% aqueous sodium chloride solution but die within 2 days at a concentration of 30%. They can live for days in 5% acetic acid and for about 3 days in vinegared soy sauce containing about the same amount of acetic acid. It has been recorded that larvae remained alive at room temperature for 20 days in distilled water and for 41 days in city water (KAWADA). Of antiseptic, antibacterial and antioxidant agents, salicylic acid and Sodium benzoate hai e some larvicidic effect. However, there are problems in their application to fish that are to be eaten uncooked (HIRAOKA (h) Anthelmintics Of various anthelmintics tole- used in man and animals, especially nematocides, thymol and tetrachloroethylene are known to be effective. However, the use of these, like food additives, for fish meat creates a variety of problems - taste, sanitation and safety regulation (HIRAOKA 2) Methods of removing Anisakis larvae from fish and shellfish In order to prevent the eating fish and shellfish infested with Anisakis larvae, it is necessary to ascertain whether parasites exist in food samples before eating. There are several methods for such a purpose and they are (1) a glass plate-pressing method, (2) a standing at.constant

21 19 temperature method, (3) an enzyme treatment method and (4) a proteaseproducing bacteria treating method. OISHI et al. (1970) compared all of these methods and have concluded that the enzyme treatment method is interfered with by the connective tissue of fish meat samples to such an extent as to make it unsatisfactory, that the glass plate-pressing nethod is the most reliable but laborious and that the standing at constant temperature method can be - accurately and easily performed on soft tissues only, such as the liver and testis. In Canada since long before the discovery of Anisakis larvae,.attempts have been made to remove parasitic nematode larvae from cod fillets in (p.610) order to eliminate consumers' repugnance to the parasite presence, and one of the methods put to practical use is candling. In this, a sample fillet is placed on a glass plate and illuminated above or below the glass plate for observation. In order to facilitate the observation under transparent light, a fish meat-slicing machine was invented and has been proven very effective in the removal of parasites. According to one report, the body of parasites fluoresces in a blue-white color under untraviolet illumination, thereby making the detection of the parasites easy. Since fish parasites are discernible to the naked eye, consumers are more sensitive to parasitic than bacterial contamination of food. Hence also in the U.S.A., food processors are taking pains in the removal of parasites from food (LISTONE et al., 1960, 1961). 3) Conclusion A variety of preventive and food sanitation measures for fish infested with Anisakis larvae have been given in the two preceding sections. In these measures several agents; e.g., concentrated solutions of

22 20 Japanese horse-radish, sodium chloride and soy sauce, salicylic acid, thymol and tetrachloroethylene, can kill the parasites, but their practical use as means of prevention and/or sanitation is difficult. The overall view is that freezing is considered to be the most promising method of prevention against anisakis infestation. Total fish catch of Japan is 10 million tons at present and a quarter is frozen. Freezing is extremely effective in maintaining the freshness and the quality of fish catch, but it is also unexpectedly useful as a means of prevention against anisakiasis. However, complete realization of practice in which all sea foods to be eaten uncooked are first frozen and then thawed to be served on the table would be difficult due to technical reasons as well as to the diversity of individual tastes. Postscript The pathogens of anisakiasis are larvae of nematodes known as anisakis and their final hosts are marine mammals such as dolphins and whales. Infestation in man (experimentally in dogs, rabbits and rats) is established by eating untreated, cold water-washed or vinegared slices of raw fish, the fish comprising more than 132 marine species such as mackerel, horse mackerel, pollack, tuna and sagittated calamary, all of which act as the paratenic hosts of the nematode. Anisakiasis is one of the recently popularized zoonoses and was first discovered in Japan in This disease is seen nationwide in fairly large numbers and many cases are considered to be overlooked. Parasitization is also observed in large numbers of dolphins, some of which succumb to anisakis parasitization. During a few recent

23 21 years, reports that were written by medical, veterinary and fisher; scientists on anisakiasis amount indeed to several of hundreds, indicating the extent of importance'and of interest in this disease. Since the Japanese like raw fish, anisakiasis is likely to occur in large numbers also in future. It should be mentioned here that the hitherto held common view that the danger of infestation with Anisa{is larvae increases with decreasing freshness of fish must be corrected, since the opposite is true. A-variety of preventive methods against anisakiasis have so far been proposed. Of these, freezing appears to be the most effective from a practical viewpoint. The author is grateful to Dr. OZURU (Niigata University Medical School), Dr. ISHIKURA (Ishikura Hospital, Iwami City, Hokkaido), Dr. YOSHIMURA (Kanazawa University Medical School), Dr. OISHI (Hokkaido University, Faculty of Fishery Science) and Dr. KAGEI (National Institute of Health) for references and advice.