Translation Series No.1414

veW/FS,

FISHERIES RESEARCH BOARD OF CANADA Translation Series No. 1414

An introduction to food hygiene of the Anisakis larva.

By Keiichi Oishi, Shigemi Oka and Seiichi Josho

Original ti tl e: Anisakisu Yochu no Shokuhin Eiseigaku Josetsu.

From: Anisakisu Yochu no Shokuhin Eiseigaku Josetsu, 1-113, 1969.

Translated bY the Translation Bureau(NO) Foreign Languages Division Department of the Secretary of State of Canada

Fisheries Research Board of Canada Biological Station Nanaimo, B.C.

1970

270 pages typescri pt J 4 /4-

DEPARTMENT OF THE SECRETARY • OF- STATE SECRÉTARIAT D'ÉTAT TRANSLATION BUREAU . •2 BUREAU DES TRADUCTIONS

é éi I FOREIGN LANGUAGES DIVISION DES LANGUES DIVISION CANADA ÉTRA NG ÈRES

TRANSLATED FROM - TRADUCTION DE INTO - . EN

Japanese English

AUTHOR - AUTEUR OISHI, Keiichi; OKA, Shigemi; JOSHO, Seiichi

■■•• TITLE IN ENGLISH - TITRE ANGLAIS An Introduction to Food Hygiene with Regard to the Anisakis Larva. Title in foreign language (transliterate foreign characters) Anisakisu Yochu no Shokuhin Eiseigaku Josetsu:

RE,F5RENCE IN FOREIGN I,ANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHA,RACTERS. REFERENCE EN LANGUE ETRANGERE (NOM DU LIVRE OU PUBLICATION), AU COMPLET.TRANSCRIRE EN CARACTERES PHONÉTIQUES.

Anisakisu Yochu no Shokuhin Eiseigaku Josetsu.

gl› REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS An Introduction to Food Hygiene with Regard to the Anisakis Larva

PUBLISH ER - EDIT EUR - PAGE NUMBERS IN ORIGINAL Hakodate Shokuhin Kagaku Kenkyukai DATE OF PUBLICATION NUMÉROS DES PAGES DANS DATE DE PUBLICATION L'ORIGINAL (Hakodate Food Science Research 1 - 113 Societ YEAR ISSUE NO. VOLUME PLACE OF PUBLICATION ANNEE NUMERO NUMBER OF TYPED PAGES LIEU DE PUBLICATION k odate Ohtani 01 0 HanaK NOMBRE DE PAGES Womenls . Junior College, 51, Kameda DACTYLOGRAPHIÉES cho Hondori, Hakodate-shigai, 1969 270 Hokkaidv.

REQUESTING DEPARTMENT Fisheries & Forestry TRANSLATION BUREAU NO. 5089 MIN ISTàRE-CLIENT NOTRE DOSSIER NO

BRANCH OR DIVISION Biological Station TRANSLATOR (INITIALS) N .0. DIRECTION OU DIVISION TRADUCTEUR (INITIALES)

PERSON flEQUESTING Mr. N.P. Boyce, Nanaimo, B.C. DATE COMPLETED DEMANDE_PAR ACHEVÉ LE May.2, 1969 '

YOUR NUMBER VOTRE DOSSIER N ° 76918-14 • On:y DATE OF REQUEST 27.1.70 DATE DE LA DEMANDE T R C NON É.0 • îç t)C.il1 s,..tizincent

503.200-10-e (REV. 2/86) ) -""t

DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT . TRANSLATION BUREAU BUREAU DES TRADUCTIONS FOREIGN LANGUAGES DIVISION DIVISION DES LANGUES ÉTRANGÈRES

CANADA

CLIENT'S NO. DEPARTMENT DIVISION/BRANCI-1 CITY N° DU CLIENT MINISTERE DIVISION/DIRECTION VILLE

769-18-14 Fisheries îf:' Forestry Biological Station Nanaimo, B.C.

BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) DATE N° DU BUREAU LANGUE TRADUCTEUR (INITIALES)

5089 Japanese 1A.O.

AN INTRODUCTION TO FOOD HYGIENE WITH REGARD TO THE ANISAKIS LARVA

Keiichi -OISHI, Shigemi OKA and Seiichi JOSHO • (Department of Food Science, Faculty of Fisheries, Hokkaido University.) (Hakodate Ohtani Women's Junior College.)

CONTENTS

Introduction 1

0 im I. Classification and Morphology of Anisakis 3 \141 e.f.e > Significance of Anisakis Classification 3 o 0 . Ï•3 0 Morphology of the Subfamily Anisakinae 4 l2 0 0 F- (1) Adult • es•E 0 *4: (2) Larva .> ? radikY,) ILJ • in LLJ ee

SOS-200-10-31 2

• Morphology of the Genus Anisakis 8 (1) Adult. (2) Larva. Summary 10 ' II. Life History and Ecology of Anisakis 12 Life History of Anisakis 12 Ecology of Anisakis 15 (1) Detection of Subfamily Anisakinae (Genus Anisakis) Larvae in Marine Pisces and Decapoda. (2) Distribution of Nematodes of the Genus Anisakis in Marine Mammals. (3) Distribution of Anisakis Larvae in Fish Bodies. I) Parasitic Conditions of the Larvae in Different Parts of Fish Bodies. In Parasitic Conditions of the Larvae in Fish Bodies of Different Ages. III) Parasitic Conditions of the Larvae for Different Fish Weights. IV) Parasitic Conditions of the Larvàe for Different Lèngths of Fish Body. (4) Monthly or Seasonal Distribution of Anisakis Larvae in Pisces. (5) Distribution of the Larvae in Pisces and Cephalopoda with Different Habitats. Summary 33

III. Anisakiasis . 35 Historical Background of Anisakiasis Research . . 35

. Epidemiology of Anisakiasis 37 (1) GeOgraphical Conditions of Outbreaks. (2) Dependence of Outbreaks on Sex. (3) Dependence of Outbreaks on Age. (4) Distribution of Gastric and Intestinal Types. (5) Summary Of. Epidemiology. Clinic and Tathology of Anisakiasis 42 Clinic. PathologY. • Etiology.

Diagnosis and Therapy of Anisakiasis 49 Diagnosis. Therapy. Summary • 54

IV. Experimehtal Anisakiasis 55 Significance of Experimental Anisakiasis 55 Mainly.Concerning the Localized Allergic Reaction 56 Mainly . Conàeiming the Conditions of.Larval Migration' into Host odies (Distribution and Detection of Larvae) Mainly COncerning the Factors of Both the Host and

Parasite Sides - 74 Mainly Concerning the Clinical State of Animals with

Anisakiasis Infection 79 4

Concerning Examples of Experiments in Other

Countries 79

Summary 80 V. Food Hygienic Countermeasures Against Anisakis Larvae , 81 Resistance of Anisakis Larvae ...... 81 (1) Resistance in Various Media. I) Salt Water. II) Bydrochloric Acid. III) Various Acids. IV) Various Alkalis. V) Various Nutriments. •VI) Various Seasonings. VII) Rearing Liquid, City Water, Distilled Water. VIII) Formalin. IX) Ethyl Alcohol. (2) Resistance to Various Temperatures. I) High Temperature. • II) Low Temperature. (3) Direct Killing Effect of Various Medicines

• Methods of Collecting Anisakis Larvae 95 Summary 96 VI. Problems Remaining in Anisakis Research 98

. . Conclusion S 99 References 100 5

INTRODUCTION 1

It cannot be denied that there is a prevailing trend for parasitic diseases to become a concern of the past in our country,. This is because new anthelmintics or specific chemotherapeutants have been developed against those parasites such as ascarids or àncylostomas whose infections were wide- . spread before the war or even a while after the war, and in addition infection prophylaxis such as the improvement . of eating habits, use of washing liquid and pervasion of chemical fertilizer, etc. have become established with reasonable results. However, in recent years parasitic diseases caused by helminths other than ascarids or ancylostomas - for instance pinworms - has increased, and the countermeasures against parasitic diseases in our country are being reviewed in a new perspective and are raising new problems in the fields of clinical medicine and public hygienics. One of these is anisakiasis which is to be described below. Although anisakiasis may be new to the layman, it has taken much attention and become a major topic among medical or parasitological societies. Although this disease has been warned against many times in newspapers (1966, 1968), it appears that the hygienic countermeasures are not enough and recognition by the general 6

population is low. It had been an established theory until recent years

at least . that a parasite whose proper hosts are originally various animals other than human beings does not have an infection ability towards man and does not demonstrate pathogenicity. Recently, the theory of visceral larva migrans proposed by Beaver et al. (1956) of the United States on the basis of research into symptoms shown when ascarids of dogs infest human bodies opened a new approach to the zoonosis problem. . That is, they demonstrated in human body experiments using the eggs of mature ascarids of dogs the fact that various kinds of helminths of mammals other than man enter the human body which is an abnormal host, and continue parasitic phenomena temporarily without becoming mature,

'? and there display varying pathogenicity and bring about clinically important diseases (fever, hepatomegaly, pneumonitis, eosinophilic leucocytes, blindness), and they also pointed out that there are many cases like these clinically. For example, it is known that 'human gnathostomiasis' or 'eosinophilic meningitis' may be caused in humans by the Consumption of freshwater fish or shellfish which are the intermediate host of Gnathostoma spinigerum in dogs and cats . and of Canton hemosite nematode in mice.

Anisakiasis is a disease caused by certain kinds of 7

larvae of the genus Anisakis, family Ascaroidea which infest marine mammals, when they enter the human body perorally through marine fish or cuttlefish, and this disease is understood to be included in the above-mentioned visceral larva migrans. The existence of anisakiasis was confirmed by Van Thiel (1960, 1962) in Holland. It was given much atten- 2 tion in Northern Eurôpe a8 a disease which occurs among peoples with the habit of eating raw herrings in Northern Europe centered on Holland. In Japan it was Keizo Asami (1964, 1965) who confirmed anisakiasis. From then, research on Anisakis.was rapidly commenced and it was found that it • has been far more prevalent in'our country where there is a strong traditional habit of eating raw fish than in Holland (Oshima, 1968). There are many parasitic diseases caused by fish or shellfish already known, such as heterophyasis including clonorchis sinensis and metagonimiasis which are caused by - the consumption of freshwater or semi-freshwater fish, and the danger of eating raw freshwater fish is well known. On the other hand, although it is acknowledged that extremely large numbers of young helminth exist in marine fish (Hoshina, 1963), only one kind of tapeworm (Diplogonoporus grandis, Japanese double cord tapeworm) was suspected of giving lesions to the human body. Tapeworms are parasitic on marine fish • or on Oncorhynchus keta or Oncorhynchus masou which originate 8

from fresh water. However, it has been reported that there is no harm in eating raw fish if they are the usual marine fish (Hoshina, 1963). Anisakis larvae, however, are parasitic on many marine fish, for example Gadus macrocephalus TILESIUS, Theragra chalcogramma (PALLAS), Enemplatopharus japonicus (HOUTTUYN), and even Ommastrephes sloani pacificus, and they cause larva migrans.. In this respect, anisakiasis has appeared as a parasitic disease which contradicts past common knowledge. •When the process of anisakiasis. research in Japan is surveyed up to the present, we whose aim is research on , the food hygiene of marine products realize the importance of this disease which is thought to result from eating raw fish (especially fish caught in the northern area centered on Hokkaido), considering it a problem significant for our own selves, and we fully realize the necessity to deal with it and consider this our mission. Although our knowledge and experience of anisakiasis is superficial, we were able to collect a hundred or so original books, references or newspaper articles with the assistance and cooperation of many people, including Mr. H. Ishikura locally in Hokkaido who is a pioneer in this field, T. Nishimura (University of Osaka City)., T. Yamaguchi (Hirosaki University), H. Yoshimura (Chiba University), • A. Kobayashi (National Institute of Health), J. Yamashita' • (Hokkaido University), personnel of Hokkaido University, 9

Department of Veterinary Medicine, Parasite Class, and many others. By knowing the progress and present position of anisakiasis research through these references, the authors are endeavouring to pursue fundamental studies in order to • devise food hygienic countermeasures against this disease. Bearing this in mind, the authors will describe in the following chapters the current understanding of anisakiasis, how to deal with it and study it food-hYgienically -, mainly by the references mentioned later, in arder to enable one to gain a general idea of anisakiasis. • 10 I. CLASSIFICATION AND MORPHOLOGY OF ANISAKIS 3

Si nificance of Anisakis Classification

The method of classification which is most generally accepted at present i8 the one by Yorke & Maplestone (1926), and according to this the taxonomical position of Anisakis is defined as follows: Phylum Nemathelminthes. Class Nematoda. Order Eunematoda. Superfamily Ascaroidea. Family Heterocheilidae. Subfamily Anisakinae. Genus Anisakis.

Including Anisakis, ten genera such as Contracaecum, Porrocaecum, Raphidascaris and Paranisakis, etc. have been established in the subfamily Anisakinae by recent research, but Hartwich (1957) modified this classification slightly by including Porrocaecum from the above-mentioned in the family Toxocaridae, Augusticaecum and Amplicaecum in the family Ascarididae and furthermore, he considèred the twO Raphidascaris and Paranisakis genera as Raphidascarinae, excluded them 11

from the subfamily Anisakinae and instead added Terranova, making them six genera in total. On the other hand, Mosgovoy (1951) proposed dividing the genus Anisakis into two subgenera, i.e. Anisakis and Skrjabinisakis, and he considered the main morphological difference between these two subgenera to be in the gastric region next tO the oesophagus. On this basis, he classified 18 species, that is, 13 species of subgenus Anisakis, 3 species of subgenus Skrjabinisakis, and 2 species of undetermined subgenus. These are shown in Table I-1 (Kagei, 1967). There is considerable dispute as to the independent characteristics in each species, for instance many people regard the three species of Anisakis . simplex, Anisakis dussumierii, Anisakis kukenthalii as one and the same species (Baylis, 1932; Lyster, 1940; Kagei 'et al., 1967), so it is presumed that the number of independent species will essentially be far less. In fact the nematodes of the genus Anisakis or subfamily Anisakinae which are found at present in the principal fish of our country are as follows, and these are further explained in Chapter II, Ecology Section. Anisakis simplex (Anisakis type I) A. Typica A. Physeteris (Anisakis type II) (Anisakis type III) r)

Terranova sp. Contracaecum sp. (Contracaecum type A) C. sp. (Contracaecum typc B) Raphidascaris Amplicaecum

Table I-1. Presently used Anisakis spenies. (Môsgovoy, 1951)

A nisakis , Dujardin, 1815') Mosgoroy, 111rd '

A. ■ A nisakis dussum ierii Beneden, 11.1101

Baylis, 1920

A. A. a lexandri II su et I Io eppl i, 1133

A. A. ratudontis Itaylis, 19119

A. ■ A.. ■liomedeae 1. instow, Miel • 11 a) lis, 1923

A. A, kokenthalii

A. A. insignis 1)iesing 1 ■.r2,1

A. A. i‘ Mo,govoy, 19-19

. A. A. . pa t agonira l.instov, 1*() IL.vli,192 3

A. A. robuirs ri Baylis, 1916 layli, 1 9;:i11

A. similis Baird, 11-11;3 Baylis, 192.9

A. • A. !,irnplex It odolphi, 11l ■9 • Ilayl s, 1 9.10

A. A. t rident at a K rois, 19:11-1

A. A. typira 1.-a;11 • Baylis, 19;10

A . Skrjahini sakis skarjal.ini Mosgovoy, 1,19

A. S. physet eris IlyIis, 111'.13

A. S.• .sdlupakori 1■10,govoy, • 1951

A. I cogians JI1t1 It"Nlaw snn, 1 , r19

A. 1 tursionis C rusr, 1941i 1 3

Morphology of Subfamily Anisakinae

(1) Adult

The principal morphological characteristics of adults of the subfamily Anisàkinae are as follows: There are three labia at the anterior extremity, which is a characteristiC of ascarids, and there ire differences between male and female. The posterior part'qf the oesophagus forms a ventriculus exhibiting a glandular structure. A single long and narrow renette cell can be observed in the left • side of the false coelom extending from the oesophagus to the intestinal canal of the worm body. However, there are some exceptions, one of which is the gastric region. There are • three genera, i.e. Dujardinia, Amplicaeéum, Angusticaecum which do not have this region. The second exception is the renette cell and Porrocaecum,

whose normal hosts are birds, lacks this cell. • , Therefore, it has been proposed that it will be appropriate to exclude these genera from Anisakinae and to . include them in Toxocaridae. The most prominent morphological differences among each genus of the subfamily Anisakinae are in the alimentary canal, especially the morphology of the gastric region and the presence or absence of a ventriculus 14

appendix or intestinal caecum, and apart from these the aperture of the renette cell is an important differentiating feature.

(2) Larva

It is also possible to distinguish larva by the characteristics of the alimentary canal in almost, the same manner as adults, but the distinguishing features are the aperture of the renette cell e presence or absence of a .oring tooth and of a mucron in the caudal region. A special question here is the morphological differences among the four genera of Anisakis, Contracaecum, Terranova and Porrocaecum which are suspected to have the ability to infect man. Such morphological characteristics were investigated by Inatomi et al. (1966) and Oshima (1967) with regard to four genera including Raphidascaris instead of Porrocaecum (Table 1-2) and by Kato et al. (1968) concerning six nematode genera of the subfamily Anisakinae which were detected in fish from Tokyo Central Market (Table I-3). Moreover, Otsuru (1969) demonstrated the morphological Characteristics of the anterior and posterior ends of nematode 'larvae of the subfamily Anisakinae by schematic diagrams as in Fig. 1-1.

IR (--D M -n• 11 r.V1 k:H b; et gl, ..t. Oesophageal Intestinal PI /.. 0") .. fig 5".. ; It. 'et, ql t l..!...c ,.„: •■ rt ei t2. PI i8( --II, appendix ri , , I 2 ) ( ) 4 , .q‘a.I :fi. by,41:tkAi csectun / t i ' i,g IaSIIP. 1 Ankskis 1 10 -36c, 0.3 -Mom - - 7.0m 17 -25 38 -50 18 4 I 60

Anisakis . 11 24 -33 05 -0.7 - - ? 233-37 50 - 72 ?

Terranova 33) 0.5 -0.7 - + ? ? ? ? ?

Raphidas-cari.1 8 -10 0.2 -•0.3 I- - 1.8 ? • 12 13 ?

Contrite:locum 15-17 0.2 -0.3 •t 1- 2 .0 ? 18 20 6

Table 1-2. Morphological differences in larval nematodes which infest fish and cuttlefish.

1. Name of genus. • 2. Length of larval body. 3. Width of larval body. 4. Thickness of cuticle. 5. Number of cells in the intestinal cana] (1 / 2). 6. Number of muscle-cell layers(1 / 4). 7. Muscle-fiber bundle. 8. Number of parallel arrangements .. 9. Number of radial muscle-fibre bundles.

0 firi. (i ',.. k, I ' czAnil cpAn "' Raphidascaris Contracaccum Porrocaecuni Terranova D -, - -4- (Boring tooth ) , ce m re _ (Inter labia t - CD D I. vi ki 7) (Ventricules ) -- -+ _ _ Ventriculus appendix: - R î * - - _ (Intestinal caccurn ) + + D fe. V 3Z' e _ ( Mucron ) + - + +

_ D e.te,5eh-lo)« _ _ + + --

28.4mre 30.3oo D. eitc,),,,è , 6.2 -17 .2rien 8.9 -15.0mul 30inntiq li. 30ntn;..1 '.1. (19-36) (24 - 5-3 - 9 0 l3-:P■ >2R'it.q)Il#lOS{i1t't- eD (Renette cell ) U(174 .)ti i%e

Table 1 - 3. Morphological characteristics of nematodes of the subfamily Anisakinae 16

1. Species. 2. Anisakis type I. 3. Anisakis type II. 4. Characteristics. 5. Boring tooth. 6. Inter labia. 7. Ventriculus. 8. Ventriculus appendix. 9. Intestinal caecum. 10. Mucron'in caudal regions. 11. Presence or absence of formation of reproductive organs. 12. Size of larval body. 13. Aperture of renette cell. 14. Long ventriculus. 15. Short ventriculus. 16. Labia. 17. About 30mm.

Ii Antsakis Ardsakis A lisakis Terranova Cont racaecum C on tracaecum II III A 1

Fig. I-1. Anterior and posterior ends of larval nematodes of the subfamily Anisakinae. 1 7

Shiraki (1969) investigated the morpholoical characteristic's in cross section of each nematode larva of the subfamily Anisakinae collected from marine fish. There is no difference in the cuticle among Anisakis types I, II, III and Terranova, but in Contracaecum type (A) larva small tubercles can be observed on both right and left sides and in type (B) the cuticle layer is thin. The lateral lines of Anisakis and Terranova larvae in a transverse cross section present what is termed a twinleaf-like .shape. In Anisakis types I and II, the two 'leaves' of the lateral line are separated -from each other, in type III and Terranova larvae they are always fused together, and in Contracaecum (A) and (B) the two 'leaves' are wider and their fusion tendency is stronger. No notable differences can be observed in the oesophagus cross section of each larva. The intestinal caecum of the Terranova larva can be observed in the cross section of the gastric region and that of Contracaecum types (A) and (B) larvae in the cross section of the posterior half of the oesophagus. The excretory gland in each larval body is contiguous to the left lateral line and tends to lean towards the sides. Apart from Contracaecum type (B) larvae, the excretory gland in each larva becomes enlarged about midway along its length, the enlarged part being between the gastric region and the anterior part of the mid-gut in type I, in types II, III and Terranova larvae it does not

enlarge until . the mid-gut region, and in Contrac.aecum type (A) it enlarges at the anterior end of the mid-gut. An opening for the excretory pore is present near the labia in each larva of Anisakis, Terranova, Contracaecum type (A), but in Contracaecum type (B) larvae it opens on the ventral surface slightly posterior to the nerve ring. Table 1-4 shows the dimensions of each part of nematode larvae of the subfamily Anisakinae which were taken from various species of marine fish.

AnisaMs Contracaceum Terranova

1 .(9) 11 ( 2) M M 09 A M B 03)

23.0-317 26 .7, 31.9 25.0, 26.3 16 .2 - 48.0 1b.-29.7 93.5 - 35.3 (28.6) . (32.6) (23 .4 I ( 28.1 )

B. W. 0.43-0.52 0.71, 0.71 0.97, 0.97 0.42-095 0.58-0.72 0.40-0.61 (0.49) ( 0.80 ) (0.64) (0 .51)

Oes. 1.76 2.3 1.80, 2.06 1.69, 1.75 1 .54 - 3.18 1.28-1.54 2.0 -2.7 • (2.1 ) ( 2.1 1 (1.38) ( 2.4 )

Vent. 1.08-1.46 0.62, 0.71 0.55, 0.52 0 .60 -1.84 0.29-0.38 (1.29) ( 1.22 ) (0.32) 0.35-1.17 052-1.02 1.8 -2.4 Int. Caec. / / / (0.69) 10.79) (2.1 )

Vent. App. 0.77-1.35 / / . / / 0.94)

Tail 6.10-0.13 0.23, 0.31 0.15. 0 .19 0.10-0.22 0.17-11.25 0.14-0.27 (0.12) 0.J4' 1 0.21) (0.20)

Table 1-4. Measured dimensions of each type of larva. * Number of larvae measured. Units are mm. Numbers in brackets are mean values. B.L.: Body length. B.W.: Body width. Oes.: Oesophagus length. Vent.: Ventriculus length. Int. Caéc.: Intestinal caecum léheth. Vent. App.: Ventriculus appendix length. Tail: Tail length. 1 9

The differentiation of these larvae is not always easy, but larvae of the genus Anisakis have neither ventriculus appendix nor intestinal caecum, Contracaecum larvae have both of these, and Terranova and Porrocaecum have intestinal caecum only. However, a distinction between the latter two is almost impassible. Moreover, it has been considered that since there is no development of an • intestinal caecum in • Porrocaecum larvae of body length less than 28 mm, it is impossible to distinguish them from Anisakis larvae by this feature (Baylis, 1944). However, with regard to this point, there have been some opposing assertions that even in larvae of shorter length (about 21 mm) an intestinal caecum can be observed (Berland, 1961; Poljansky, 1955; Scott, 1955; Kobayashi, 1967). Furthermore, there are some larvae of different genera which are considered able to enter the human body via marine fish and which are morphologically very similar ta the larvae of the genus Anisakis. One of the principal of these is Eustoma.. The final hosts of this genus are sharks and rays and it is possible to distinguish this genus from Anisakis larvae, because in the latter the excretory pare of the renette cell is near the labia and anal glands are present, whereas larvae of the genus Eustoma lack anal , 'glands and the excretory pore is near the nerve ring. 20

Morphology of the Genus Anisakis

(1) Adult

The characteristics of adults of the genus Anisakis are the various features.of the subfamily Anisakinae and in addition, the excretory gland aperture is situated near the . labia, they lack an intestinal caecum and a ventriculus appendix, they do not have interlabia but have dentigerous ridges and.their cross section exhibits two-leaf shaped or Y-shaped lateral lines. There is a morphological difference between the subgenera of Anisakis in the ventriculuS, the subgenus Anisakis having a long ventriculus and the subgenus Skrjabinisakis a short one. Morphological features which are points for differentiation among speCies are generally more conspicuàus. in males than females, especially the number . of post-anal papillae and the ratio of the length of the right and left spicules, etc.. In female adults, the position of the vulva is one of. the identification features, but this is not sufficient for differentiating from other species. Kagei et al. (1966) investigated dolphins, whose distribution density is the highest of all the marine mammals inhabiting our coast, and examined the adults of the genus Anisakis which were detected in the stomach. The result

was that they were mostly Anisakis simplex and Anisakis typica, and some immature Anisakis were also observed. Anisakis causing granulomatosis in our people are presumed to be mainly the two types A. simplex and • A. typica, according to Oshima (1966) and Kagei (1966). The • morphological features of these three adult species including It_lnyseteris are demonstrated by Kobayashi (1967, Table I-5). Moreover, there is a similar report in the investigation of nematodes of the genus Anisakis parasitic on marine mammals conducted earlier by Kagei et al. (1967, Table I-6).

TJ It le fr( ...".."-...-7 ' It / Ilr r 1 1 t Mlle • oll; ç''''' ■■ d 37 - 133 i 0.9 - 2.5 S '-"FILk '.;) ? rI 191 • : 4i t L i Gu

■ :`:. - 3 / 7 79-200 4 2.7 - 2.75 2 --,-- .: i E L a -'0.n) s ,-. 1 •f-'4. 2 4 3.7 A. aomplex 60-120 d 1.2 -2.5 1.6 -3.5 4;1.6 -2.6 ;IQ' I /2 O' I 95-140 4. 2.3 -3.5 61 (ii) : 1.5 I/ 2.06- 1 / 2.5

6+0.96 40 1 d 31-70' d 1.0 -1.5 S96 CD 1011 (a k 3. illi''Pfk 27 A. 4. 37-90 7 1.5 -2.0 , 5- I:. 'FLO 9 - 1171 G) 1 : 2-9

533 ( i k 0. 4 . I(10.35 C2) 30% d 70 -90 d 3.0 I , '1' ( ('' ) I II ' 3" ei ei 11 irw, e oe._ 2 / 9 -S, 80 -112 4,0 - 4 , "i . 3 'PAU ' 11.911) A.lerteri., ›; L 05 I / 4 - 1 / 5.5 d 3 -4 S 100 -145 1.0 4 1.1 4 3 ,1 CP, a .1,,,iii,le, q- 130 -200 3.5 -5.0 0,8 - ) . 5 671

Table I-5. Morphological comparison of three species of adult Anisakis. 1. Species. 2. Body length(mm). 3. Body width(mm). 4. Gastric region(mm). 5. Sigmoid. 6. long lengthwise. 7. Straight. 8. Longitudinally and laterally almost equal. 9. Number of post-anal papillae. • 10. Seven pairs (One pair of them combined papilla).

11. 6 - 8 pairs. 12. 6 pairs. 13. 10 pairs. 14. 9 - 11 pairs. 15. 6 pairs (One pair of them combined papilla). 16. 4 pairs. 17. 6 pairs. 18. Length of right and left spicules (mm) or ratio.

- 19. Both right and left 1.68. 20. The two are not equal, left 2.4 - 3.7, right 1.6 - 2.6. 21. Left 3, right 0.96. 22. Left 0.4, right 0 -.35. 23. The two are*almost equal in length, 0.33 - 0.5. 24. The two are almost equally short. 25. Position of vulva from anterior end. 26. Approximately 1/2. .27. Approximately in the middle.

' - il 0 t n t o) ; - -: ,,!. ! v; 3;: v) • co rej‘. 'e:-- 1 ci...,5• • • .4. simplex 65 .n ;,1 ,',D. f".3 i!,_ :,< ,,.0 P:, i • 1 : 1.5 A. typica 90 rr. Y+ ,, ' Ill f , .. ry. :',0 F .• 10 1 : 2.9 e.C . ,

.),-,f'.1,L,,,,,.,I, .,, A, physeleris .. • ',.: ( 'if. ; .', ' '*; e. 30 '.'..; 100 .0.. ,....: „ 0

• 5 t e„. ■kn '.,. 1.,

Table 1-6. Major morphological differences of Anisakis found in various marine mammals. 1. Species. 2. Size of adult. 3. Colour of body. 4. White-yellow. 5. Brown or pale brown. 6. Shape of ventriculus. 7. Rectangular. 8. Approximately square. 9. Number of post-anal papillae (pairs). 10. Ratio of length of right and left spicules. 11. The two are almost equally short. 12. Position of vulva.

13. One pair is • combined papilla.

14. Anterior end - Vulva length • Body length 2 3

The larvae of the genus Anisakis which are found in various fish and Ommastrephes sloani pacificus on the Japanese coast may be divided into two broad types (long ventriculus type and short ventriculus type). The long ventriculus type larvae correspond to the type I and the short >ventriculus type to the type II proposed by Berland (1961). Oshima and Kagèi et al. (1966) pointed out that if the genus Anisakis are divided into two subgenera, the long ventriculus type (type 1) conform to the subgenera Anisakis in adults and the short ventriculus type (type II) to the subgenera Skrjabinisakis in adults. According to the measurements by Koyama et al. (1966), the body length of type I larvae is 19 to 36 mm (average 28.4 mm), body width 0.26 to 0.58 mm (0.45 mm), They have a distinct boring tooth in the limpàss and a renette cell runs . longitudinally in the left of the body cavity, with its aperture between the ventral labia. The oesophagus is divided into muscular and glandular portions (ventricular region) -and the cross section of the lateral line exhibits a tWo-leaf shape. The morphology of type II is almost identical to that of type I. The points of difference are that in type I, the ventriculus is long (body length / -ventriculus = 25.5) and the joint between the ventriculus and intestinal canal is oblique, while in type II, the ventriculus 24

is . short (body length / ventriculus = 49.0) and the ventriculus joint is flat. Moreover, there are some other differences such as that type I has a mucron in the caudal end, but there is no mucron in type II. Furthermore, it was reported that A. simeex and A. typica develop from type I in the third larval stage and A. physeteris from type II in the saine , stage (Kagei, Oshima, 1967). However, it is difficult to maintain that the taxonomical research of Anisakis larvae is already established as there are different opinions among researchers. That.is, according to Koyama et al. (1966), these larvae are divided morphologically into several species, and according to Sakumoto et al. (1966) there are four • species which parasitize Trachurus japonicus alone. Moreover, Nishimura (1966) holds the opinion that there are more than four species according to the morphology of helminth bodies which passed into the stomach and the enteric canal of experimental animais, especially according to the shape of the dentigerous ridges, but Oshima (1966) and Kobayashi (1967) consider them . as two species of types I and II. On the other hand, Otsuru et al. (1968) recently recorded a type III which. has a short ventriculus and tail, from fish (Theragra chalcogramma, Conger myriaster) in the northern Japan Sea, and divided Anisakis larvae into three types •• .(refer to Fig. I-1). Moreover, Ishikura'et al. (1967) found that the

2 5

Anisakis larvae discovered in intestinal Anisakis disease affected parts and the larvae which parasitize Theragra chalcogramma both belong to the same type I. That is, they made a morphological comparison of Anisakis larvae found in intestinal wall and from Theragra chalcogramma and human showed the measured values of several parts (Table 1-7). Similar examination results are published in the reports by Yoshimura (1966) and by Kumada (1967), and these are all shoWn together in Table I-7.

' . ; a •.,K A _®. ÇD__: ;e 0 a% ieràb Exam Mt, -Max --..% Marl.-Max. -, : Min..-Max. ',1-.-. Min. -14 ... 9-..0 M.." --M. ' rE "

T 7-.. 1 ,:53 15 0-.96.5 29.4 5.26-0 . 59 0,45 2.34-4.54 3 . 36 0.65-1.50 1.12 0.08 -. 0.16 0 . 12 Fg...nt 52 13 3-33.0 2:. 4 0,24-0,55 5 40 1 50 -3.15 2.54 0 55-1.00 0.74 0.08-0.16 0 .12 ..'./..8' 87 '15664 1 24 3 0.51 3.32 1.19 0.11 0.12 .1 ..... . e, . ■ 1 27.5 0.47 3.06 0.76 •'. 1 1 , ::-....a) 1 0.56 0.1 10.87 " I 0.51 21 .1755 1.07 1 0.5 0.1 a. 1 0.55 0.12

Tree II :5 19.0-25.5 27.8 0 55-0.70 0.61 2 10-3.42 2.73 0.43-0.75 0.57 0.18-0.32 0.25 1 25.4 0 67 2.22 0.52 0.18

0 7, ■ 1968) 6

Table I-7. Comparison of Anisakis larvae collected from the human body with those from marine fish. 1. Body length. 2. Average. 3. Body width. 4. Oesophagus. 5. Ventriculus. 6. Tail. 7. Human cases. Yoshimura (1966). Kumada (1967) Ishikura (1968) 8. By Oshima (1968). 26

As a result, the larvae parasitic on Irl_e_nErp.... chalcogramma and the larvae, although with imperfect bodies, found in the human body exhibited approximately similar values. Therefore, it can be said that, aithough there were few human examples, these results provide important data for the purpose of analyzing the mutual relation between Anisakis larvae type I which infest Theragra chalcogramma and human anisakiasis. Teramoto (1966) gave several points as clear morphological characteristics to enable the differentiation of Anisakis larvae from human ascarids.

SUmmary

In the above, we described the taxonomiCal position and the morphological differences or characteristics of the larvae and adults of the subfamily Anisakinae (genus Anisakis) as a pathogenic organ of anisakiasis, mainly by referring to the literature of KobaYashi (1967). However, when reviewing the process in the past, opinions among researchers regarding the classification and the identification of the larvae based on their morphology 'are not necessarily the same. Nevertheless, the establishment of a method of identifying the larvae of subfamily 27

Anisakinae (genus Anisakis) which are pathogenic organs of anisakiasis is urgently needed by the food hygienic countermeasures in our country and it is an important problem which cannot be neglected. It is very difficult to identify Anisakis larvae merely by minutely observing their surface centering on the alimentary canal as a measure of identification. Therefore, it seems that the morphological identifying tech- nique of larval body transverse cross sections proposed by Koyama et al. (1966) and by Oshima et al. (1967) will be a promising method of differentiation for th â future. The common opinion that the pathogenic organ of anisakiasis is the type I larvae Fily not be absolutely correct. Moreover, we must give serious consideration to the possibility that other close genera belonging to the subfamily such as Terranova, Contracaecum, Porrocaecum and Raphidascaris, etc. cause Anisakis-like disease. The reader may think that the authors used too much space for the classification and morphology of Anisakis. However, this subject has many yet to be solved problems for the future and it is a basic part of food hygienic research against AnisakiS larvae. Therefore, we must be fully aware of this subject at present, and at the same time we should have enough knowledge to be able to deal readily with the ‘subject, however it might change in the future. Therefore, we spent this amount of space with the view that we should put at least this much weight on it. 28

IL LIFE HISTORY AND ECOLOGY OF ANISAKIS 12

Life History of Anisakis

Kobayashi (1967) and Kagei et al. (1968) give the following account: The final hosts of nematodes of the genus Anisakis are cetaceans or pinnipeds. Taking Anisakis simplex or Anisakis typica as examples, the principal final hosts are cetaceans, especially dolphins, and they are parasitic in the ingluvies. The adults are white-yellow, the body length being about 60 mm. The ova produced from the female are expelled into the sea with the host's excrement. The cells inside the ovum divide and develop into a larva. This larva hatches out by itself and swims free in the sea water. According to experiments by Kobayashi et al. (1966), the ova hatch in two days at 27°C and 11 days at 2°C. Moreover,.Kagei (1969) observed that the sea water temperature is an important factor when the ovum develops into a larva inside the egg shell, not only for A. simplex but also for ova of any of the subfamily Anisakinae (Table II-1). If the sea water temperature is slightly higher than 10°C, development is more rapid, and in temperature higher than 27°C growth is even faster, but they degenerate and die 2 9

in mid growth. At low temperature, for instance at 2°C, the larvae grow if reared for a long time and can be observed to hatch out and move around actively in the sea water. The length'of larvae newly hatched into sea water is about 230 to 280p, their width being 14/x. , the first stage larvae are encysted and they possess a boring tooth in the head area Growth proceeds through the first, second, third, fourth stages and at the fifth stage the larvae become adults. One can only make presumptions about subsequent growth; but referring to the report by Poljansky (1955), it is thought that small Crustacea such as Euphausia become the first intermediate host and they develop to second stage larvae in the same host body (Oshima, 1966). More recently, Oshima et al. found Anisakis-like larvae in Euphausia or other planktons collected by people on board "Osho Maru", a train- ing ship of Hokkaido University, in the North Pacific Ocean from June to August, 1968. These investigation results will . be published fairly soon. Moreover, early in the same year, Oshima et al, conducted infection experiments on Euphausia on the ship "Todai Tansei Maru". It was observed that when the larvae were in contact with Euphausia for two hours, they were taken into the Euphausia body as is shown in Table II-2, and most of them moulted about one.hour after . infection (Oshima, 1969). The second stage larvae which parasitize Euphausia enter the bodies of fish or Mollusca

:,.. re, 1:- • . -', (t) t21 0 111311 "e2; . Ter.lr.ova 2 - 4 t 4 1 '18D.lejeerr i"Uell,1.1 71 ,.') Scott, 1955 d,,,p.,Is n - m 3 5 7 13 -14 17 -25 2 3 4 el, 8 - 9 lin2-3 91M -t. (i.t)

7 0 .,7...... /.:.rum n _ _ _ 25 -30 FilliZinger, 1966 2 1 1 - 2 3 3 - 4 5 - 7 _ Anteçuke, 2 30 60 ■ , ?tilde, ' - 5 - 40 •1'11- - q c,, 1966' 27 1--? 2 - 3 3- 4 7? yf-,b. :_ k i., ( q.1) 1968 (il . f . 1

Table II-1. Time (number of days) requir'ed for the growth of nematode ova of each genus of the subfamily Anisakinae at various temperatures. 1. Species. 2. Temperature. 3. Morula stage. 4. Gastrula stage. 5. Larval stage. 6. Free swimming. 7. Source. 8. 4 days. 9. (There was no further growth under observation up td 18 days.) 10. (Died after two to three hours) 11. No growth. 12. Degenerated without growth. 13. Degenerated withôut swimming free. 14. Kobayashi, Kagei et al., 1966, 1968.

C) eel k 13 e eVe 1. it;'...: .' 5,'

_ 0 % '13.7,, hat &ilk - - % 286.1 1, M a (262.2 -342.6 ) (12.3- 15.3)

D 1 F 4 50 4.5 22.2 237.6 13.9 (245.5 -318.0 ) (13.1-14,8) 259.7 b 20 5 20 2.0 50.0 14.5 (254.4 -265.0 ) (13.6-15.3) _ - 0 0 - 231.4 17.8 D 6 50. 2.3 85.7 (187.6 -266.9 ) (12.9-20.6 4-80 )'

Table 11-2. Infection experiment on Euphausia and morphological changes of the.larvae. 31

1. Number of days after infection. 2. Soon after hatching. 3. 7—day. • 4. 2-days. 5. 3 days. 6. 4.to 8 days. 7. Waller of Euphausia examined. 8. Infected Euphausia. 9. Number of larvae per Euphausia. 10. Number of molted larvae. 11. Overall length of larvae. 12. Width of larvae. 13. Method of fixation. 14. Heat. 15. Alcohol. Formalin.

Fig. II-1. The life cycle of Anisakis 1. cetaceans, dolphins, pinnipeds (fourth stage larvae and adults). 2. Helminth ova. 3. First stage larvae. 4. Second stage larvae. 5.11211.!ML11.2.- 32

6. Pneumatophorus japonicus 7. TFd--(Theragra chalcogramma) 8. Trachurus japonicus 9. Cololabis saira 10. Ommastrephes sloani pacificus 11. Third stage larvae.

Contracaecum Cont racaecum Terranova Anisakis aduncum snicul igerum decipiens simplex sea water EGG' EGG • EGG ECG

1-st, LARV.A 1-5t. II-ARVA 1-st. LARVA 1-st. LARVA

i(1) IC) • 2-st. LARVA 2-st. LARVA 2-st. LARVA 2-st. LARVA

I, int. host. Jr 2-st. LARVA . 2-st. LARVA 2-st. LARVA 9-st. LARVA

/ s . . Invertebrate Plankton Fish Copepoda Invertebrate (Mysids Shrimp ) (Euphausiid )

2: int. host. 2-s t. LARVA 2-st. 1 ARVA 2-st. LARVA 2-st. LARVA

small Fish•,,,, small

3-st. LARVA 3-st. LARVA 3-st. LARVA 3-st. LARVA

Def. host. 3-st. LARVA 3-st. LARVA 3-st. LARVA 3-st. LARVA

Fish Bird I Seal j Dolphin 1 (•!)

4-st. I ARVA 4-st. I ARVA 4-st. LARVA 4-st. LARVA

(3 I@ • 5-st. 5-st. ( ADULT) 5-st.(ADULT) 5-st. (ADULT ) (ADULT) (Berla rul , (Holzinger. 1566) (S cott , 1954, 55, (Kagei, 1968) m yers. 1960)

Fig. 11-2. Schematic diagram of the life history of subfamily Anisakinae nematodes. ŒD: Molting (numerals indicate the order of molting). st-: Stage. 1. Source 33

and become third stage larvae. It is considered that, when the fish body with the third stage larvae becomes the prey of the final host Marine mammal, the larvae penetrate under- neath the .mucous membrane of the final host stomach with their head_section, and there they enter final parasitic life and becoMe adults after two more moltings. Man also becomes infected by the third stage larvae through fish or cuttlefish, but since man cannot be their final host, they die without becoming mature soon after inVading the alimentary canal wall. Kagei (1968) presented an outline of this at the symposium "Concerning anisakiasis". The abovementioned prodess is shown abbreviated (Fig. II-1). Moreover, Kagei also reported the life history of other nematodes of the subfamily Anisakinae in a schematic diagram (Fig. II-2).

Ecology of Anisakis

It appears that there are not many reports on the systematic investigation and distribution of the subfamily ' Anisakinae or genus Anisakis larvae. However, within this limited knowledge, we wish to discuss the distribution of larvae and adults of Anisakis and closely related nematodes, 34

and detection observations. The following items are arranged for this purpose.

(1) Détection of Subfamily Anisakinae .(Genus Anisakis) Larvae in Marine Pisces and Decapoda

According to an investigation by Sana (1966) concerning the distribution of Anisakis larvae, the percentage of Pneumalophorus japonicus (off the coast of Sanriku) infested was 75.5%, averaging 30 parasitic larvae per fish; 19.7%, averaging 3 larvae, for Trachurus japonicus (off the coast of Sanriku) and 55.4% for Clupea pallasi (Hokkaido). Moreover, Yamaguchi (1966) reported that when he examined 1,400 fish of 40 marine species obtained in Shikoku district, these larvae were found in 23 species of fish such as Pseudorhombus cinnamomeus, Paralichthys olivac eus, Pagrus major, Pristipomoides filamentosus, Seriola quinqueradiata, Katsuwonus pelamis, Thunnus thynnus, etc.. When Nishimura (1965) conducted a similar investigation on 13 species of fish sold in Osaka district markets, the percentage parasitized was 66.6% in Trachurus japonicus and 33.0% in Pneumatophorus japonicus, and a maximum number of larvae per fish of 438 was found. Later, Okumura (1967) investigated 30 species of fish and 4 species of cuttlefish and detected

these larvae in 13 species, i.e. 12 fish species and 1 species of cuttlefish, and the detection percentages were between

20 and 90%, although it varied within fish species(Table II-

3 ).

3) % IA -Dew 5)%1I se allz . ,7 238 218 92,0 2,388 10.9 GD V 7' :',.:- 103 93 90.2 3.683 39.6 (D e. 4 160 114 71,1 1,338 11.7 m f 27 16 59.2 132 8.2 f., 4 5 26 14 53.8 114 8.1 le 1- '.> 71- 24 19 79.1 2,42 7 127.4 -= '.% :4 39 34 87.1 175 5.1 ..X.71-'7Y.5 23 16 69.5 253 15.3 ' 7 f 5 11 3 27.3 29 9.7 4 y- 4 11 7 63.6 26 3.7 )7t7V74 4 3 75.0 6 2.0 5 3 60.0 5 1.7 ' t ," 4 el 277 207 74.7 287 1.4

0 e m118tau481amnl_e

Table II-3. Parasitic conditions of Anisakis larvae in Pisces and Decapoda. 1. Species. 2. Number of fish examined. 3. Number of positive fish. 4. Percentage positive. 5. Total number of parasitic larvae. 6. Mean number of parasitic.larvae. 7. Pneumatophorus japonicus 8. Trachurus japonicus 9. Dentex tumifrons 10. Platycephalus indicus. 11. Coryphaena hippurus 12. Trichturus lepturus 13. Clupea pallasi 14. Theragra chalcogramma 36

15. Gadus macrocephalus 16. Pagrus major 17. Branchiostegus japonicus japonicus 18. Hippoglossoides dubius 19. Ommastrephes sloani pacificus 20. Number of parasitic larvae per positive fish or

On the other hand, the results of an investigation on marine fish in the coastal waters of Niigata and Hokkaido conducted by Otsuru . (1968) between 1964 and 1965 were as shown in Tables 11-4,5. According to this investigation, the larvae were found in 14 out of 32 fish. species obtained in the Japan Sea near the Niigata coast, and the mean number of larvae per fish was more than five in the four species, Theragra chalcogramma, Pneumatophorus japonicus, Katsuwonus pelamis and Clupea pallasi. Moreover, Anisakis larvae were detected in 12 out of 18 fish species in the coastal waters of Hokkaido, Terranova larvae were found in Gadus macrocephalus, Hippoglossoides dubius, Stichaeus grigorjewi, Sebastolobus macrochir, Osmerus dentex and Hypomesus olidus, and Contracaecum larvae were found in Theragra chalcogramma. Moreover, Kobayashi et al. (1966, Table 11-6) investigated thè infection of Anisakis larval nematodes in 20 species of fish and cuttlefish centered on Japanese coastal waters and found 16 species to be infected. He reported that among these both the infection percentage and number of

parasitic larvae were high especially in Theragra chalcogramma, Gradus macrocephalus, Oncorhynchus masou, Katsuwonus pelamis, Lampris regius, Pneumatophorus.japonicus, CluPea pallasi and Ommastrephes sloani pacificus.

a) * 41 ‘.. tletintrc (%) fee **ti 1)o) MIUSYSIS • U 8.21_0E. RI ( ) tt

g* 4, ,... -, , 1.253 /1.332( 95 ) 8.488 (14) ■ 2.616 11 118 (14) 8.3 10 /10 (103 ) 82 (39)••• 82 (39) 8.2 0 7.7. Flee 73 /73 (100 ) 3.126 49 3.175 43.5 1) -= *), z••• .10 /12 ( 83 ) 58 6 64 5.3 1 '7 5- 2 /27 ( 7 ) 2 2 0.07 ›. 21 /22 ( 95 ) 53 53 2.4 ..., "7 2/3 ( 67 ) 3 3 1.0 .7 1., • -a 3 /21 ( 14 1 6 . 6 0.3 - 7 7 f., 11 /86 ( 13 1 20 1 21 • 0.24 9, , 1 2 /10 ( 20 ) 2 2 0.2 g)rii - eet..4 7/36 ( 19 ) 30 30 . ' 0.8 ) » /- Y./ s• , , 2 /3 ( 67 ) 2 2 0.7 e -I- .7 e 1 /6 ( 17 ) 1 1 0.2 93.7. ri, X 1 ' 8 23 /148 ( 16 ) 43 43 0.29

Table II-4. Results of investigation on Anisakis larvae in marine fish and cuttlefish. (March 1964 to September 1967, caught mainly in the coastal waters off Niigata) 1. Fish species. 2. Number of positive fish / Number of fish examined (%) 3. Number of larvae detected. Anisakis larvae type I (type II). 4. Visceral organs, Abdominal cavity. 5. Musculature. 6. Total. 7. Mean number per fish body. 8. Pneumatophorus japonicus 9. Katsuwonus pelamis 10. Theragra chalcogramma - 11. Clupea pallasi 12. Oncorhynchus keta 38

13. Oncorhxnchus masou 14. Pleurogrammus azonus 15. Cololabis saira 16. Trachurus japonicue • 17. Paralichthys olivaceus 18. Tanakius kitaharai 19. Lepidotrigla microptera 20. Trichiurus lepturus 21. _LmasOn - Iessloanieficus * Some of these were caught off the coast of Sanriku. ** Caught offshore from Sanriku. • (Note) The following fish were all negative. Numerals in brackets •are the number of fish examined. Seriola quinqueradiata (15), Sillago sihama (36), Prognichthys agoo (6), Sardinops melanosticta (5), • Arctoscopus japonicus (5), Arrosomt_gz rentattts (5), Pagrus major (3), Branchiotegus japonicus (3), Talus tumifrons (2), Clupanodon punctatus (23), Thysanophrys spinosus (3), Lateolabrax ;Iaponicus (3), •Navodon modestus (3), Hemiramphus sajori (2), Watasenia scintillans (254), Metapenaeopsis barbat'a (52), Penaeus japonicus (10).

(ji) Ca.• ititen-,‘ TPA ( ) ein t).(41/i es 90103 ) Anisakis , Terranova Callracaecusa D ak tat 1 Fitts% efttr.c fritlia eamstt Vittit /31±srt 9 318 X .7 >cf.> lo 10 669 . (2) - - - _ 7 - 7 A ff v 4. 26 12 . 27 5 -. _ - _ - • e ,50 i .1 e - - _ 99 7 8 8 8(3) 9 n' 9 './ 9.'/I . - _ 4 4 3 3(1) .566. Deus'. al2FIftetittftn --, L•YàRle) .;. - ir . .5 2.1 II - 138 2 . 6 :-, a 1.7.71.'/e9 .à à 397 , . -_ _ _ .7. .0: . ..e 11 à à _ _ Is . I I _ je -.... ef.0 . 0 _ 2 - i s. t a / - / v 4 ' 4 0 0 - I 7 -9 •e w i. 4 8 ' - ,7'7•st,/i• 4 .' 11 . - 11 t• 4 ' (0 '2 - - - - lo 5 19 _ _ - . 12 1 1 r - t. .?> .•./. 5 :9 7 5 11 ,: . - 4 1 2 - — i. .-3 - 4 a. 1 1 1 0 'I .7. A 19 1 _ •‘. ' 7 •,' V 10 o 0 - - - _ e 10 0 0 :_ -

Table II-5. Results of an investigation into larvae of the genus Anisakis or closely related genera in the marine fish of thè . coastal waters of Hokkaido.

(July to October, 1967) 1. (a. Investigation of the total fish body. Numerals in brackets are the number of larvae detected in muscle.) 2. Fish species. 3. Number of fish examined. . 4. Number of positive fish. › . 5. Number of larvae detected. 6. Theragra èhalcogramma 7. HiPPoglossoides dubius . 8. Sebastolobus macrochir 9. Osmerus dentex 10. Hypomesus olidus 11. (b. Investigation of visceral organs, abdominal . cavity and abdominal wall) 12. Gadus macrocephalus 13. Theragra chalcogramma 14. Acanthopsetta sp. 15. Tanakius kitaharai

16. Hippoglossus stenolepis 17. Microstomus achne 18 - If.2112.EELIIA_michn11 19. Cleisthenes sinetorum herzensteini 20. Clidoderma asperrimum 21. Astroconzlranyriaster 22. Stichaeus nozawai 23. Stichaeus grlanlewi 24. Sebastolobug macrochir 25. Osmerus dentex 26. Arctoscopus japonicus 27. Osmeridae sp.

Anisakis ot ex t 4it e % e. Contra- Raphia- Poro- TA caecum ascaris caecum

) . 5- 7 5 100 % 36.2 40% 0 % M% e....41 ti v 1 5 7 100 13.3 0 0 7 Pl- 2 5 7 X 10 100 20.0 70 0 0 ti 71, '..-• e •.:, t 1 100 9.0 0 0 0 T 2 100 76.5 0 0 ci t 5 't ,c 30 83 6.8 0.03 0 0 'MUii•it.i.i•d'fflieQ a :." 41 71 4.5 66 0 0 lt..,"e 1• .."*. WC2.3 v 'I' ., 20 55 2.5 0 0.05 0 . 1Pu-tei5i) OE il •:/ * 12 33 1.3 0 0 0 T 131 7 7. f.:, 82 15 1.8 11 32 0 Tr4 • ,),Egm - mf...ee V 20 5 1.0 0 0 o cgb v 70 20 0 0 0 0 0 cj D4 % tl 4 13 0 0 0 0 0 c?› 7 4 7 ,., 20 • o 0 0 0 0 10 0 0 0 0 0 1:1 h(e)

x R.. X 4 h 101 50 3.3 0.05 0.01 0

1 ) 4 h 24 0 0 0 0 0 1 , 4 ,, 23 0 0 0 0 0 ,.. thIcià 20 0 0 0 0 0 it5 ZO

• ( ) . Ut- (1967) I:

Table 11-6. Parasitic conditions of larval nematodes of the subfamily Anisakinae in various marine Pisces and Decapoda. 41

1. Fish species and the number examined 2. Percentage infested. 3. Mean number of parasitic larvae. 4. Placé caught. 5. 211.1f.1.2.1q1.2.2EIMITI 6. GadUs macroceEhalus 7. Oncorhynchus masou 8. Katsuwonus Pelamis. 9. Lampris regius 10. Pneumatophorus japonicus 11. Clupea pallasi ' 12. Pneumatophorus tapeinocephalus 13. Sarda orientalis 14. Trachurus japonicus. 15. Cololabis saira Thunnus thynnus 17. Oplegnathus punctatus 18. Sardinops melanosticta 19. Limanda yokohamae 20. Oinmastrephes 21. Doryteuthis bleeker 22. Idiosepius sp. 23. Sepia esculenta • 24. Decapoda sp. 25. Hokkaido. 26. Shimoda. 27. Off the coast of Africa. 28. Kamaishi, Hokkaido, Odawara. 29. Hokkaido, Ibaragi. 30. Izu-hichito. • 31. Shimoda. 32. Shimonoseki, Odawara, Boso. 33. Off the coast of Sanriku. 34. Joban. • 42

35. Tateyama. 36. Chiba. 37. Fukushima. 38. Hokkaido, Niigata, Nagasaki. 39. Mie. 40. Ibaragi. 41. Kochi. 42. Off the coast of Africa. 43. By Kagei (1967).

Moreover, Yamaguchi et al. (1968) also investigated the infection of Anisakis larvae in 1,850 fish of 53 various marine fish species . obtained in Hirosaki City, and they detected Anisakis larvae type I in 15 species such as Clupea pallasi, Pleurogrammus azonus, §.22rnber_Imonints, etc. and among them the infection percentage was high in Pleurogrammus azonus, Theragra chalcogramma, Gadus macrocePhalus, Oncorhynchus masou, Pagrus major. They also examined Doryteuthis and

Oiuinastrephes sloani pacificus and detected larvae in 168(25,8%) out

of 660 Ommastrehess_ an cificus . Contracaecum larvae were detected in Theragra chalcogramma and Trachurus japonicus,' Amplicaecum larvae in Trachurus japonicus and Terranova larvae'in Gadus macrocephalus. Orihara et al. (1968) also conducted an investigation of fish collected in the coastal waters of Hokkaido and stated that Anisakis larvae were found in Theragra chalcogramma and Orama.s - ficu.s_ ,

Contracaecum larvae in Theragra chalcogramma and Terranova larvae in Ommastrephes sloani pacificus. • • 43 Kato et al. (1968) also investigated the distribution of nematodes of the subfamily Anisakinae in 1,540 fish of 70 marine fiSh species reaching Tokyo Central Market, and they reported that the overall percentage of species in which larvae were detected was 78.5% (55 fish species), the percentage detected with larvae (types I and II) of the genus Anisakis was 64.3% (45 fish species), that with Anisakis. type I larvae was 60% (42 fish species), that with type II was 24.2% (17 fish species), and the percentage detected with the genùs Raphidascaris was 18.6% (13 fish species) and that with the . genus Contracaecum was 20% (14 fish species). Moreover, according to this report the detection percentage for type I genus Anisakis larvae in the common fish, Scomber japonicus, was 89%, the highest percentage after Theragra chalcogramma (95%). Abe et al. (1968) also investigated the infection of genus Anisakis larvae in 79 fish species, 3 Decapoda species, 1 Octopoda species and 1 species of • shellfish during the period between January 8, 1968 and the end of March of the same year and they reported that the infection percentage was especially high in 14 fish species such as Gadus macrocephalus, Theragra chalcogramma, Scomber japonicus, Katsuwonus pelamis, Lateolabrax Imonicus, Branchiostegus japonicus japonicus, Decapterus russelli, etc.. Moreover, Shiraki (1969) collected Anisakis and closely related larval nematodes from a total of approximately 44

1,500 of 15 Pisces and 2 Cephalopoda species caught in the coastal waters off Tohoku and Hokkaido districts from 1966 to 1968, and investigated whether they had tissue invading habits (Table II-7). The fish species parasitized by these larvae and the capture locations are shown . below. Anisakis type I larvae: Gadus macrocephalus, Theragra chalcogramma, Clupea Pallas', Oncorhynchus . keta, Oncorhynchus masou, Pneumatophorus l_aponicus, Katsuwonus pelamis, Tracb1.1112_1aLl_rLialli, Arctoscopus japonicus, Ommastrephes sloani pacificus (Throughout the waters investigated, Table II-7). Anisakis type II larvae: Pneumatophorus japonicus, Katsuwonus pelamis (Off the coast of Sanriku). Anisakis type_III larvae: Theragra chalcogramma, Konger myriaster (Off the coast of Kushiro). Terranova larvae: Gadus macrocephalus, HippoglossoideS dubius e Stichaeus grigorjewi, Osmerus dentex,

1.12£2171.22-112-12_, Als.122_2.2na.s_1222n12n2- (Off the coasts of Otaru, Kushiro and Wakkanai). Contracaecum type A larvae: Theragra chalcogramma, Gadus macrocephalus, Osmerus dentex (Off the coasts of Niigata, Otaru, Kushiro and Wakkanai). Contracaecum type B larvae: Ommastrephes sloani pacificus (Off the coasts of Niigata and Kushiro).

fr z.., 0 -U.Iv'e.n. , 9i i/ 4. 1JE iit t.iLe.m

Anisakis 8./.) I , iî) 4-, -...., -et WI A) '66. 11-'67. 7 114 /207 55.1 1.82 0 v 3' 5 ( hi F1) '68. 3 2 / 5 40 1.6 qD it. › -v, ,, (4*.k. 83) '66. 10-267. 11 15 / 19 78.9 1.58 Q..) ;I, i / .« (E. F1) '66. 12, '67. 4 77 /127 60.6 1.45 I. r> , 5 (ell' A) '67. 5 4 / 35 ' 11.4 0.11 ‘.DI :--' !., i (± M) '68. 3 5 / 90 5.5 0.08 ® 7 8 8 1.., 4 (+ M) '68. 3 2 / 28 7.1 0.07 0.03 7 ( VI g4) '67. 5 1 / 36 2.8 CC) # . r> 9 '7 21- MI Pi) '68.3 2 / 95 2.1 0.02 .71) 4- . (d , M) M. 3 1 / 50 2.0 0.02 ) Terranova î.)') 0 v Y" 5 ( hi Pi) '68. 3 5 / 5 100 4.8 (TD .. ,. e (VI eé) '67. 12 29 /259 11.2 0.12 # . r> 9 r) 21- (41. Pi) '68. 3 11 / 95 11.6 0.12 7 8 el- 4 (+ M) '68. 3 1 / 28 3.6 0.04

(a) #- . •-; 1) '74. (' b 44) '68. 3 1 / 50 2.0 0.02 )Contracaecum Mi.t.,* 8

IL .), 4 I) Oh A) '66.8, '67.6 22 /154 14.3 0.23 0) .7. 11.• Y 4 8 ( DI 1$ ) '68.9 2 / 20 10.0 0.15

Table II-7. Parasitic percentage of each larva inside musculature and the number of parasitic larvae. Oct. 1966 to Sept. 1968 1. Fish species. 2. Location caught. 3. Anisakis type I larvae 4. Pneumatophorus japonicus (Niigata) 5. Gadus macrocephalus (Wakkanai) 6. Pneumatophorus japonicus (Akita) 7. Pneumatophorus japonicus (Sanriku) 8.. Theragra chalcogramma (Niigata) 9. Clupea pallasi (Otaru) 10. Hippoglossoides dubius (Otaru) 11. Trachurus japonicus (Niigata) 12. Osmerus dentex (Wakkanai) 13. Osmerus,dentex (Otaru) 46

1. Fish species. 2. Location caught. 14. Terranova larvae 15. Gadus macropephalus (Wakkanai) 16. Arctoscopus japonicus (Kushiro) 17. Osmerus dentex (Wakkanai) 18. Hippoglossoides dubius (Otaru) 19. - Osmerus dentex (Otaru) 20. Contracaecum type B larvae 21. OmmastrePhes sloani pacificus (Niigata) 22. Ommastrephes sloani pacificus (Kushiro) 23. Time investigated. 24. Number of fish infested / Number of fish examined. 25. Mean number of parasitic larvae.

Among these larvae, only the three species of Anisakis type I, Terranova and Contracaecum type B were parasitic in the musculature. Anisakis types II and III and Contracaecum type A larvae were only detected in the abdominal cavity or on the surface of visceral organs. Of Anisakis larvae found in Pisces, three types are known at present. Type I larvae are found in many fish, and in our coastal waters it has been reported that 47 families, about 80 species of Pisces and 1 species of Mollusca, Ommastrephessloani pacificus are infested by these larvae. There are some reports from abroad, and if we include them, more than 100 species of Pisces and Mollusca are intermediate . hosts of this larvae. On the other hand, type II larvae had only been found in the abdominal cavity of Lampris regius 47

and Katsuwonus pelamis, but recently they were also found in Katsuwonus pelamis caught off the coast of Sanriku and in prcn.etheirisp_roneLis (Oshima. 1967) caught in the fairly deep waters of Sagami Bay and in PneumatoEhorus japonicus (Otsuru et al. 1967) landed in Niigata. Anisakis type III larvae were also found in Kushiro. From the above information it is çonsidered that the main nematodes of the . subfamily Anisakinae (genus Anisakis) found in Pisces and Cephalopoda in Japanese coastal waters are Anisakis types I, II and III, Contracaecum, Terranova and Raphidascaris. The above reports mainly concerned the distribution of Anisakis larvae in marine fish, concentrating on Japanese coastal waters.

In the 1957 to 1958 investigation reports of the International North Pacific Fisheries Commission of Japan, the United States and Canada, it is stated that about 50% of the salmon caught in the North Pacific to the west of the longitude 180 0 east line are infected by more than 25 Anisakis larvae (probably type I) per fish. It was reported, however, that those caught on the eastern side, Canada and the States,

are only slightly infected or not infected at all (Oshima, 1968). . Moreover, investigations on freshwater or semi- ' freshwater fish have been undertaken in our country, and Otsuru et al.. (1967) detected.Anisakis type I larvae in 48

Tribolodon taczanowskii among 7fish species caught in the Akanogawa and Shosen rivers and pointed out that marine fish are not the only intermediate hosts of this larVa. It is worth noting that the danger of infection by Anisakis also exists in semi-freshwater fish.

(2) Distribution of Nematodes of the Genus Anisakis in Marine Mammals

With respect to the parasitism of nematodes of the genus Anisakis in marine mammals, these nematodes were at first reported as the genus Ascaris by Jagerskiold (1894), and Stiles and Hassal (1899), etc.. After the taxonomical and morpho],ogical basis of nematodes of this genus was established by Baylis (1920), a large number of reports were published such as those by Baylis (1932), Johnston & Mawson (1945), Dollfus (1948) and Margolis (1954), etc.. Table I1-8 shows the Anisakis nematodes in various marine mammals as published by the above àuthors up to the present. The reported habitats of the mammals, their presumed populations and food habits (Nishiwaki, 1965) are also shown in this Table.

21 l ® • I.b 0 .!s 't a v til t ri ro- -1; It ti -• -is i L1,-77 rin T 4 4. A h .7 iil.e-4 : n, ,K ai 4 e. (5-.'D 4 ti■De) It

I; 47 . . e7 r 7 -9 •1• 111 t1 IA 1.000 4- 1 Cl) + r * .7, I IA 1.003 - 1.500 500 20.003-13.000 0, 4 '7 ... 7 .., 5 + + + 4 7 t • !I. tA 1.000 + 50,003 -7(0) e + ' • --; -., e ..1 7 r 4 7" 7 • *SA 20,000 50.030 0.■ , 9 ,,,, , 1- ,1.11tV0 5.000-7.030 + 1.030 10.000-15,000 , ••- 41.3 7 ••• 5 + .7- * -,• 1 • 01171 15.000 + 5.0)0 ejV 4 n + 7 .9 , .% 9 + + + :A • le .t t.. 70. 0))-100.000 .1. + 4 4-IA a + + • aft* 7 4 + 4 eta • + 7 • e e , e n e e• 9 4 + •• 4 31 11.14113 + + c) te.-7...,) + 7 +

+ +nrii.an* .5140 - 3.0)0 &)4, 4-e + 4 77 Ill • 4 me, .000 -3.003 ',Of.," ■ -I- le.ein .0011 - 10,030 + •Ii.•Pcnbgp 4- n»,..., r-evrnv4.77 ? (5' . r•ev,..1 :439 .1 / ■• h 4 + CIA* ' €(). +. 30,030 + 4 ...• F,V••É. ? + 4 :•• F.71...C. 4 77 !A + + + •••••1,1n -efiloh + 4 n SA • *SA + VA)* . + 150 . CO3 -200,003 + + r r • * 77 + + 4 771A • NIA 7 + r 1' , • • r•• h 4 .• • ,1 (L.- •• • -i h I lk r, 4 ? + < ...• r .$ "...D4 = 5...... • .7 9 4 .I .. 4 ••• n. • + + 50.00 . 4 . I f3 4 77 IA • 74 .4)14 (3)' 30.003 -50,003 • 7 + 77 IA • n :Oh 4- • + 4V 1.4 ,...... • • z • 1 , + entUATMAI * + 4 + 4 , I- .1 (•. -, 9 &• r• + *IA • 4 ntA • 3, 703 -50,000 + + + + 4 1111 50.000 (10.000-20.07X] (:) • 411414 + ■ • 71.•510 0ii-•••• I y 5. y* + I (gD *474- 4574 • *IA + t . I I . t .i h t . , I. • + + + 7 + r r • .7 • 1 1 1* )1 • «7e) , • ... 4-. • 4 crt1t5. 100,010 -1710,04• r •5 • e • • * • +in 1 4 1 7 e , 1 7 n6: ut ■ ng1(1 ider1.1111. ...„. • • • •• , 9 9 •• *IA , QD" t, D • •.. P V' 7 , I • .7 • , eve ... 7 1

I 11 •• (1 14•7• I 7 c)

Table 11-8. Anisakis nematodes found in various marine mamMals to date, and the relevant food habits and inhabiting conditions of the mammals. 1. Host name. 2. Food habit 3. Cetaceans. 4. Eubalaena glacialis Small crustaceans. 5. Balaengptera musculus Euphausiacea. 6. Balaenoptera acuto-rostrata Euphausiacea, Pisces. 7. Balaenoptera borealis Euphausiacea, Pisces. 8. Megaptera nova-eangliae Planktonic crustaceans. 9. Balaenoptera physalus Euphausiacea, Pisces. 10. Physeter catodon Decapoda, Benthic fish. 11. Kogia breviceps Decapoda. 12. Ziphius cavirostris Decapoda, Benthic fish. 13. Ziphius sp. Decapoda, Others. 50

1. Host name. 2. Food habit 3. Cetaceans. 14. Meso.prodon bidens 15. Berardius bairdii Decapoda, Benthic fish. 16. Monodon monoceros Decapoda, Others. 17. pelphinapterus leucus Benthic fish. 18. - Fishes in mud. 19. Inia geoffrensis 20. de1 j i is Gregarious fish. 21. Delphinus capensis 22. Decapoda. 23. Lagenorhynchus albirostris Decapoda, Pisces. 24. Tursious_Irmas Gregarious fish. 25. Stenella caeruleo-albus Decapoda, Pisces. 26. Steno bredanensis 27. Stenella longirostris kunitom6i 28. Phocaena phocoena Herring, cod. 29. Phocaenoides dallii . Decapoda, Gregarious fish. 30. Globicephala melaena Decapoda, Gregarious fish. 31. Pseudorca crassidens Decapoda, Gregarious fish. 32. Pisces, Decapoda. 32. Globicephala sp. Decapoda. 34. Pinnipeds. 35. Eumetopias jubata Widely ranged, mainly fish. 36._ Zalophus sp. Mollusca, Pisces. 37. ArctrocephaIus australis 38. Otaria 39. Odobenus rosmarus Bivalves. 40. Hydrurga leptonyx Penguins, Others. 41. Phoca vitulina Decapoda, Octopoda, Pisces, bivalves. 42. Pusa caspica 43. Mirounga leonina 51

L Host name. 2. Food habit 34. Pinnipeds. 44. Mirounga angustirostris_ Pisces, 45. Halichoerus grypus 46. Phoca sp. 47. Habitats and presumed populations. 48. North Pacific. 49. Sauth Pacific. 50. North Atlantic. 51. South Atlantic. 52. Antarctic. . 53. Arctic. 54. Others. 55. River Ganges. 56. River Amazon. 57. Indian Ocean. 58. Caspian Sea. 59. By Kagei et al. (1967).

On the other hand, there are only a few reports, by Yamaguti (1941, 1942) and by Kikuchi et al. (1967), on the taxonomy and morphology of the genus Anisakis nematodes which infest marine madmals in the coastal waters around our country. Kagei et al. (1967) carried out an investigation of the parasitic conditions and a morphological examination of the genus Anisakis nematodes in the five families, seven genera, seven species of marine mammals which mostly inhabit the North Pacific centered on the offshore waters of Sanriku and migrate to the Izu Peninsula region from November to January every year and are caught there, that is, in 411 Stenella caeruleo-a1bus, 132 Phocaenoides do1lii which inhabit the northern waters, 28 Phocaena phocaena, 1 De1phinapterus leucas, 1 Globicephala sp., 2 ____à___ternPhrse - s and 52

1 Callorhinus ursinus. Tables 11-9,10 show results of investigations on infection by genus Anisakis nematodes, mainly in dolphins. As a result, Anisakis type I larvae were found in all the mammals examined, and type II larvae were found mainly in physeter macrocephalus. Moreover, concerning adults, the three species A. typica, A. simplex 22 it_Ehyseteris were found, among which A. simplex were observed to be parasitic in all the dolphins, their numbers being especially high in Stenella caeruleo-albus. Actypiça were found in Stenella caeruleo-albus and Phocaenoides dollii, and A. physeteris were found in no mammals other than nyseter macrocephalus. In addition, Yamashita et al. (1967) and Machida (1967) investigated Eumetopias jubata (sea lion), Phoca vitulina (common seal) and Callorhinus ursinus (fur seal), and their results are also included in Table II-10. If the above reports are summarized, the genus Anisakis nematodes which parasitize marine mammals are gen- . erally more prevalent in cetaceans, particularly in dolphins, than in pinnipeds, and regarding the species, A. simplex is the most common followed'by Actuiça and A. physeteris. It is concluded that the nematodes of the genus Anisakis in the coastal waters of Japan are mainly the three species, A. siMpleX, A. typica which infest dolphins— 23 • mostly Stenella caeruleo-albus, and A. physeteris which parasitize Physeter macrocephalus.

1 '„ :ie. 4.. jEj LT.:MI- 4 Anisakis :...q-z IL, ; 4 J1, M ( ."-0 ) 1) 1' .... :-.% 4 It-77 e 0 19.3544) 10,113 6 4 4 (190.0 ) 19.651:= 10M 250 15 9 ( 60.0 ) a 1965e. 11)1 19 10 9 ( 90.0 ) ri 1965 11fl 68 52 43 ( 82.7 ) a 1965 1f- 11M 24 8 15 9 ( 60.0 ) a 19654-- 11M 27 8 194 139 ( 71.6 ) ;Ill.- (1PU4--.)e 4). (),J, it 2.c,0 2n ( 73.4 )

19564- 12/1 28 121 41 ( 33.9 ) /11?:■.- (iPa (- A.r.,) 0 It 411 254 ( 61.8 ) ) .i, X 6 4 it, e 196,6 6/1 - 711 29 9 ( 32.1 ) -' 7 ',...&Ç

C 4 > 4 IL, 7, 19664 6)i-711 132 11 ( 8.7 ) -,.. - 9 '., 7

0 'a (1967)

Table II-9. Infectivity studies of genus Anisakis nematodes in dolphins of the coastal waters of Japan. 1. Date investigated. 2. Number of dolphins investigated. 3. Number of dolphins infected by Anisakis, 4. Location dolphins captured. 5. Stenella caeruleo-albus 6. Year. 7. Month. 8. Day. 9.. Sub-total. 10. Total. 11. Phocaena £hocoena • 12. Phocaenoides dollii 13. Futo (Izu Peninsula). 14. Kawana (Izu Peninsula) 15. Bering Sea. 16. By Kagei et al. (1967).

7' . 1 :r1 à 9 u-)••=to-,.;.",' ee qk , Stqf se..AI tR',; t L'1.1.• T-' 1 ri physeteris ,s3) . te. typiess 1 i C.) .., ) 4 , w n 411 61.8 47 0 sa . sr:z t !.s., b. 4 ,-'-, (1967) 35 _--- 9 , ,,,-;;, -7X. ,.. 4m.n 28 nA 59 o n (1967) , , 4 i..• 1) 1.52 8.7 72 0 28 " it (1967) v , 7 5 3 1(0.0 10 39 23 1tA ,I1 .'t , (1967) 0 , 3 IceJm I o 110 WI' s', ( ( 067) r, , 1967) I , , -7 .! 5 , 1 0 0 0 0 (I) — r., , ■ 1 t'fi 00.0 0 0 55 "El- n3 (1967) _ 4 es (1_..08)

Table II-10. Results of investigations on infection of genus Anisakis nematodes in mammals of the Japanese coastal waters. 1. Species. 2. Number of mammals investigated. 3. Anisakis infection percentage. 4. Number of parasites per mammal. 5. simplex or typica 6. Type I larvae. 7. Capture location and source. 8. Izu Peninsula, from Kagei et al.(1967) • 9. Bering Sea, II 10. Coastal waters of Hokkaido," 11. tt , from Yamashita et al. • (1967). 12. • , from Machida (1967). 13. By Kagei (1968).; 14. Stenella caeruleo-albus 15. Phocaena phocoena 16. Phocaenoides dollii 17. Physeter catodon 18. Eumetopias jubata 19. Phoca hispida 20. Callorhinus •rsinus 55

(3) Distribution of Anisakis Larvae in Fish Bodies

The distribution of Anisakis larvae in fish bodies has also been investigated and reported by almost all the

researchers listed in Section (1) above. Here, according to the reports of these researchers, we will describe the parasitic conditions of these larvae by classifying fish according to the body parts infested, age, weight and body length.

1) Parasitic conditions of the larvae in different parts of fish bodies

The actual state of the infection conditions of these larvae was roughly explained in Section (1). That is, these larvae generally exist in the form of a cyst or in the free state in the various visceral organs, muscles or in the abdominal cavity of fish bodies. When this is observed more carefully, it is discovered that the parasitic conditions vary according to the different intermediate hosts.

According to the reports to date (Kobayashi, Kagei, 1966; Okumura, 1967; Otsuru, 1968; Kato et al., 1968), it has been generally acknowledged that the places of predilection of Anisakis larvae are the abdominal cavity or the 56

visceral organs rather than in the muscles, except for some fish.species. Some of the observation results which became the basis of this conclusion have already been shown in Tables 11-4,5, but here Tables 11-11,12 which are results of an investigation by Kato et al. (1968), are added. Furthermore, according to Okumura (1967) it.was observed that there are variations among fish species not only in the parts parasitized by the larvae but also in the cyst formation conditions and larval body length as shown in Table 11-13. For instance, in Pneumatophorus japonicus, each . cyst was formed by a single larva mostly, and the . 110 shape resembled an annulus, while.in Trachurus japonicus , more than half of the cysts were compound type, circular in shape, and in the extremely large cysts, they were formed by an assembly of up to 18 larvae and even if small the cysts e. were normally formed by two or three . larvae. The cysts in Theragra chalcogramma and Gadus macrocephalus were the single type, circular in shape. 57

(1 C) ® 40 38 95 36 t 95 2 (5.3 Ou ) 62 :50.0"o ) C) -7- 142 124 87 120 ( 97 =- 117 55 47 52 ( 95 5 (9.1 'o) 7 j- 26 12 46 12 (100 0 111 32 29 27 - ( 84 7 (22'u) 0 G7 h 1, 4 26 6 23 6 (100 37 13 37 (10 ) 0 a .? 275 * h. X h 36 4 11 4 :100 1 ( ) 't 7 90 6 '6.7 6 (100 0 0 (37 h h 7 X 62 • 4 6.5 4 ((90 . )

( ) Pi tril'mteci. ,1

Table II-11. Parasitic conditions of Anisakis type I larvae in :different parts cf fish as observed in the main fish species. 1. Species. 2. Items. 3. Number of fish investigated. 4. Number of fish infected. 5. Percentage infested %. 6. Items. 7. Visceral organs. 8. Musculature. 9. Theragra chalcogramma 10. Scomber japonicus 11. Clupea pallasi 12. Conger myriaster 13. Ommastrephes sloani pacificus

• 14. Hippoglossoides dubius 15. Trachurus japonicus 16. Stichaeus grigorjewi 17. Cololabis saira 18. Sphyraena obtusata . 19. Note: Brackets show values as a percentage of the number of fish infected. 58

® c) . L. ®I& Fl z i 0 - E.à - it. (Z)

® I .410 396 (96.6r,, , 81 (19.8 e n) 42ft: if 9

a Ii 42 26 (61.9%) 16 (-ILLY, ) 17.'t e

Table II-12. Detection percentage in different parts (Anisakis type I and II larvae) 1. Types. 2. Items. 3. Number of fish possessing larvae. 4. Items. 5. Visceral organs. 6. Musculature. 7. Remarks. 8. Type I. 9. Type II. 10. Fish species.

Moreover, he reported that concerning the larval body length, the larvae which parasitize Pnel._.1Lma:.jpi.s'aoni.cus and Ommastrephes sloani pacificus are the largest, the mean larval body length in Pneumatophorus jaPonicus being 29mm, maximum length 33mm, and the mean larval body length in Dentex tumifrons is 19mm and maximum length 27mm. These are interesting observations not seen in any other reports. Okumura (1967) also reported the following results. Since the percentage of Pneuma tohols'a2_ .or_licus parasitized in the muscle by these larvae is high compared with other fish species, this was considered seriously from the food hygienic 'point Of view and the percentage of fish infested in the muscle by these larvae, their distribution in the variou Table 11-13. Characteristics of larval bodies found in 'various Pisces and Cephalopoda

i Fish s -cecies Mean Mean body Activity Main Shape of cyst number of length parasitized parasitic (Minimum - regions larvae Maximum)

Trachurus 676 23mm not mesentry, compound(many), japonicus - (16 - 26mm) active pyloric region, single(few), fatty tissue, circular in retina, shape, 1 cyst around the cloaca, formed by 18 • genital glands, larvae(extreme liver, example)„ abdominal cavity. - Pneumatophorus 109 29mm fairly almost same as single(donut- japonicus (20 - 33mm) active above (also shape), mostly found in muscle). distorted. Theragra 46 25mm active almost the same single(mostly chalcogramma (17 - 32mm) as in Trachurus circular in laponicus shape). Trichiurus 376 21mm fairly as above compound(many), lepturus (14 - 26mm) active (also found in single(few),. muscle). many are dis- •• torted. • Dentex 44 19mm not almost the same compound tumifrons (11 - 27mm) active as in Trachurus (moderately japonicus many), mostly distorted. .

continued on page 60. • Fish species Mean Mean body Activity Main ShaPe of cyst number of length parasitized parasitic (Yinimum - regions larvae Maximum

Clupea 52 24mm fairly almost the same single(many), .pallasi (16 - 32mm) active as in Trachurus compound(few), japonicus. mostly circular in shape.

Gadus - 24 !I it single(circular macrocephalus in shape, some • distorted), Coryphaena 48 - u as above single (mostly. hippurus (also found in distorted). the intestinal canal wall)

Platycephalus 14 !, almost the same single(many), indicus as in Trachurus compound(few), japonicus. mostly circular in shape. Ommastrephes 19 25mm active many are in single (mostly sloani pacificus (15 - 33mm) secretory project- circular in ing parts of shape). the caecal pouch and vein wall, and also found in muscle.

• 61

parts and the motivation for entering the muscle were examined. The results of detecting parasitic larvae in 25 various sPecimens of Pneumatophorus japonicus and the larval distribution in the muscle are shown in Table 11-14, and in Fig, II - 3. As a result the ratio of the number of larval bodies in the abdominal cavity to that in the musculature was 7 to 3, and with regard to the parasitized parts • 26 of the musculature, 95% of the larvae, i.e. 324, were in the abdominal muscle surrounding the alimentary canal. •

e:5;tAWO 3.2 ck, A ' 41 e@ 0.16 - k 0 0.3 e) 5 t ■D 0.02

1.2

i.,;5q3■ «) 95.3% q'i5 eC) 0.061 (i C J) 5 to 4.76*0 e o1 5 lt

Fig. 11-3. Distribution of larvae in the musculature of Pneumatophorus japonicus. 1. Percentage of the invading larvae. 2. Mean number of larvae. 3. Larvae. 4. One o means five larval bodies.

0 Q edeluw819, 54 4 4 0 51-11162 (5) 55 II 1; 0 Nu 19 )4 f, re me q2 Ahr911 e 56 2 2 0 A BCD 57 5 4 1 3 3 0 2 58 1 2 0 59 G 5 1 2 2 2 0 60 35 9 6 3 29cm 3 0 0 0 61 22 12 10 1 62 7 5 2 0 4 0 0 63 6 5 I 5 0 0 0 61 20 12 8 I 30 65 5 3 2 6 0 0 0 66 22 71 1 7 • 1 1 0 67 20 12 6 8 o o 0 68 19 14 5 -31 69 7 4 3 9 1 1 0 70 17 13 4 10 0 0 0 71 20 13 7 72 7 5 2 11 1 0 I 1 73 3 3 0 12 0 0 0 74 11 11 0' 13 3 . 2 2 5 75 11 9 2 2 14 32 4 4 0 76 36 1 1 0 15 1 1 0 77 9 8 1 16 +.1 4 19 15 78 16 15 I 17 0 0 0 79 19 16 3 18 I 0 1 1 80 15 10 5 1 19 8 2 6 51 81 9 9 0 20 4 4 0 82 8 5 3 21 33 6 6 0 83 11 11 0 22 8 3 5 5 81 11 10 1 23 1 1 0 85 4 4 0 24 4 4 0 86 12 I I 1 25 I 1 0 87 .24 :8 6 26 9 9 0 M 2 1 1 27 22 5 17 1 15 1 89 '.5 7 8 28 4 0 4 1 90 37 :8 15 3 29 12 5 7 7 91 11 5 6 30 7 5 2 2 92 7 7 0 31 5 4 1 1 93 16 15 1 32 15 9 6 6 94 30 16 14 I 33 12 12 ' 0 95 23 71 2 34 4 4 0 96 6 5 1 6 6 0 3534 97 6 3 3 36 7 6 1 1 ' 95 38 19 16 3 37 6 5 1 1 99 29 21 5 2 38 4 3 3 100 10 5 5 39 6 5 1 1 101 17 611 40 8 8 0 41 4 4 0 102 39 36 22 :4 42 10 9 1 1 43 . 11 10 1 1

44 la 9 5 I 4 103 44 32 12 4C 45 76 21 5 5 104 139 40 69 46 10 8 2 2 .71 12 •-..1 47 8 6 2 2 105 11 48 7 4 3 3 S.):' 340 4 49 35 6 5 Ur.l.:47 I 1 © à I 50 24 19 5 5 • o 70.4 29 6 3 : 3 1.2 51 1 I 0 52 12 10 2 2 53 23 20 3 3

Table II-14. Results of detecting parasitic larvae in various specimens of Pneumatophorus japonicus: 1. Length of fish body. 2. Total number of larvae. 3. Number of larvae in the abdominal cavity. 4. Number of larvae in the musculature. 5. Regions of distribution of the larvae in the musculature. 6. Total. 7. Actual number. 6 3

Okumura - (1967) considers two possibilities for the migration of the Anisakis larvae into the musculature (invading motivation). One is that some of the larvae in the abdominal cavity move into the muscles as the fish grows. The other is that since it takes quite a while for the fish to reach markets after being captured, the larvae migrate into the musculature'during that time. First, larvae detection results according to fish body growth, which is considered to be .one of the above causes, are shown in Table 11-15. According .to this, in fish

bodies of length between 29 and 34cm it appears that the percentage of larvae in the muscle out of the total number in the fish body increases as the fish body gets longer, but it decreases in fish of length greater than 35cm. As a result, he mentions that the increase of the fish body length is not necessarily proportional to that of the number of larvae in the musculature. However, since the percentage . 27 in bodies between 37 and 40cm long is equal to or greater than the average and the actual number increases according to the increase of body length, we should probably interpret the above as that the number of parasitic larvae increases as the fish body length increases. • Next, the experimental results concerning the relation to the freshness of the fish body are summarized • in Table 1I-16. The freshness of Pneumatophorus japonicus

is judged by the pH of the fish meat, by the precipitation reaction of the protein and by the amount of volatile basic nitrogen, and although it was attempted to clarify the cause of larval migration into the musculature by a relation between the freshness of fish and the number of larvae detected, no dependence between them was found. Here, when judging the freshnesà, there is also the question as to whether the degree of freshness and the time passed after being captured are proportional to each other as mentioned above.

.'it'. -.(b '4, ek et:f.. (De e r, e et') l51 P1 (cm) fbe e ffi-ee %

29 - 31 16 36 29 80.6 7 19.4 32 - 33 16 72 53 73.6 19 26.4 34 18 157 107 68.2 50 31.8 35 • 22 239 187 78.2 52 21.8 38 24 279 219 78.5 - 60 21.5 37 - 38 20 287 203 70.7 84 29.3 39 -- 40 4 210 106 50.5 104 49.5

cs, It 120 1,280 904 70.6 376 29.4

Table 1 1-15. Larvae detection results in Pneumatophorus japonicus of different body length. 1. Fish body length (cm). 2. Number of fish examined. 3. Total number of larvae detected. 4. •Number of larvae detected in different parts. 5. In the abdominal cavity. 6. In the musculature. 7. Actual number, 8. Total. 65

o ® 0 -T e 1: xvi. F-,-.• Pli (D if '.itilià Na A 8 _ 13.2 1 2 (Me Ah) 5.6 (mg/100 g) 20.4 (mg/ n ) 2 8 ( u ) 5.8 i ± 3 14 ( u ) 5.8 - - 15.1 (mg/ 0 ) 4 1 ( u ) 5.8 - 15.1 (mg/ u ) _ 8.4' (mg/ n ) 5 5 ( u ) 5.9 - _ _ 6.7 g 6 1 ( u ) 5.9 (mg/ ) _ 7 5 (A1t • A'Ah)() 6.2 12.3 (mg/ u ) 12.3 (mg/ 8 3 (a Ah) (D 5.9 - - ,, ) _ _ 9 3 (. u ) 6.0 13.4 (mg/ n ) _ n ) 10 2 ( il ) 6.2. 14.7 (mg/ 11 11 ( u ) 6.3 - 5.6 (mg/ li ) _ 12 0 (e11111)0 5.9 _ 16.0 (mg/ u ) 13 0 ( u 9) 6.0 - 14.0 (mg/ if ) 14 0 ( u 7) 6.1 - 9.3 (mg/ u ) 15 0 ( u 11) 6.2 - 23.5 (mg/ n )

• (e. 17_ )

Table II-16. Relation between the number of larvae in the musculature of Pneumatophorus japonicus and the freshness of the fish meat. 1. Fish body No. 2. Number of larvae detected in the musculature. 3. Precipitation reaction of protein. 4. Amount of volatile basic nitrogen. 5. (ventral musculature). 6. (ventral and dorsal musculature). 7. (abdominal cavity) • 8. By microdiffusion analysis method.

H) Parasitic conditions of the larvae in fish bodies • of different ages

The number of larvae infecting per year was calculated in the investigation into the larval infection conditions in fish bodies of different ages by Oshima (1968, Table II-17), 'mur . and the values were mostly from one to six. It can be seen 66

that the percentage parasitized increases with increasing age. Kagei (1968, 1969, Table II-18) summarized the relation between Anisakis type I larvae in various fish and the age and food habits of these fish as follows: The amount of infection Increases with increasing fish age. However, in Cololabià saira which eats only zooplanktons, the infection does not occur until the age of four years, and in Trachurus japonicus the food habit changes to eating fish at the age of two years, which is when the larvae are • first found. Polyphagous Gadus macrocephalus and Oncorhynchus masou are strongly infected even at an early age. It is seen from the above that there is a relation between fish age and food habits which affects the percentage parasitized by Anisakis larvae.

® t•

' fb 1i 1 4- r..1 tu'e,PZ ,?) 0 ,-, h- -2 7 ?" 5 6103 1 5/ 5) 36.2 6.0 2 - 3 4 1W ( 2/ 2) 31.0 5 Y 2 3 - 4 4. 100 (10/10) 20.0 5 - 7 @ V 7' 3 5 4 100 ( 7/ 7) 13.3 3.8 0 7: ..-- 7i-- 4 g- 100 ( 1/ 1) 8.0 2.0 6) c 5 -1- 2* 83 (25/30) 6.8 3.4

,.., .. 4 - 5 71 (29:41) 4.5 1.0 0 7- n..- 14 » 1 -g- 50 (51.100) 3.3 3.3 13 7 7 .../ /: 2* 55 (11/20) 2.5 1.3 0.9 ,a V 7 '..::- 2* 15 (12/82) 1.7 Ci).-, n. •,., 1- 1.-f_ 25 ( 3/.12) 1.0 (,;■ -7 --, -:-, 34 5 (5/1W 1.0 0.3 n n

-e Th .n 1 rA 1 - (ea) 5 2- 3-1 40 1 4 /10) 39.0 13 -20 h '7" -., 6 4 ao ( 4/ 5) 4.5 0.8 O c 5 2* 46 (14/30) 3.9 2.0 4-5 2 ( 1/41) 2.0 0.4 -0.5 • D• 2* 15 1 3/20) 1.6 • 0.8 ,t) Â ), y h 24 (12/51) 1.5 1.5

e 'e,iV•k"e L)

is?) 7 h 2 / 2 46 CD h '1 e 1 1 C9 2 / 2

Table 11-17. Parasitic density (only positive examples) of Anisakis larvae. 1. Anisakis type 1 larvae 2. Whole fish body. 3. Fish names. 4. Presumed age. 5. Percentage parasitized (%). 6. Number of larvae per positive fish. 7. Number of larvae infecting per year. 8. Years. 9. Theragra chalcogramma 10. Lampris regius 11. Oncorhynshus masou 12. Gadus macrocephalus 13. Katsuwonus pelamis 14. Pneumatophorus japonicus •

15. Clupea pallasi 16. sloani • 17. Pneumatophorus tapeinocephalus 18. Trachurus japonicus 19. Sarda orientalis 20. Cololabis saira • 21. 'Parasitized in the musculature. 22. Percentage parasitized in the musculature (%) 23. Anisakis type II larvae (in the abdominal cavity only, no examples in the musculature).

„, 0 ;„ I 2 4 • 5 6 7 8 -te

.7 >-. .., rj 10 ) ..,. . 1.8 2.4 3.4 . r, i.:'ILiAt.e1CD ,-) 14 2.5 i', fi."?. ( 2 T 1 Q ) g (i ■ ), , I ., f;.'.) f, ,I. 0, . ) 5' 7 13 3 35.9 94.1 ..1 ii 1 . Q1 ) .e. •7 , 7 ; 29.9 /0.1 ift à II 0 •I .7 5 '7 y 200 :g fi 11 gl - Table 11-18. Infection conditions of Anisakis type I larvae in various fish of different ages (mean number of parasitic larvae per fish). 1. Fish species. 2. Cololabis saira 3. Clupea pallasi 4. Trachurus ;aponicus 5. Scomber dmoni2us 6. Gadus macrocephalus 7. Theragra chalcogramma 8. Oncorhynchus masou 9. Years of age. 10. .Food habits. 11. Zooplanktons. 12. Mainly crustaceans. 13. Fish (from two years of age) 14. Fish. 15. Polyphagia.

III) Parasitic conditions of the larvae for different fish weights.

Otsuru (1968) investigated the relation between fish body weight and the percentage parasitized . by Anisakis larvae from 1964 to 1967 (Table I1-19). According to this 29 investigation, the number of larvae detected per fish increases with increasing fish body weight, the average being 8.3 larvae. Koyanagi (1967) also examined the Anisakis larvae in 27 fish species mostly from the coastal waters of Japan between 1964 and 1967 (Table 11-20). In those results it was shown that the number of parasitic larvae in Pneumatophorus laponicul of body weight 200 to 400 gramsvas 2.5 on average, and for weights greater than one kilogram the average was 16.1 larvae, so that the number of larvae detected increases with increasing fish body weight. •

10 1 ( g )Qe:ze ..1•15. 1.,* (00) cy.ele ®i °I i13.14çrz 9 a>T- e-./

200-399 39 22 (56.4) 77 12 2.5 400--599 347 302 (86.5) 1,832 35 5.3 600--799 719 681 (94.7) 6,054 78 8.5 800--999 178 178 (100.0) 2,366 80 13.3 1,000 --1,300 29 29 (100.0') 468 62 16.1

(4D st 1.312 1,212 (92.3) 10,797 so 8.3

Table 11-19. Results of examination of Anisakis larvae in Pneumatophorus japonicus for different weights of fish body. (Mar. 1964 - Feb. 1967) 1. Weight (g). 5. Maximum. 2. Number examined. 6. Average per fish body. 3. Number infected (%). 7. Total. 4. Number of larvae detected.

:I (i) 1 g. ( g ) 1',« 12 rk '0 ri 11. ;5'. ( '‘, ) ® ri:

200 - 399 39 22 ( 56.4 ) 97 0 12 2.5 400 - 599 343 298 ( 86.9 ) 1,826 0 35 5.3 600 - 799 713 675 ( 94.7 ) 6,029 0 78 8.5 800 - 999 178 178 (100.0 ) • 2,366 1 80 13.3 1,000 -1,300 29 29 (100.0 ) 468 1 62 16.1 . 0 14 1,302 1.202 ( 92.3 ) 10,786 0 80 8.3

Table 11-20. Results of detection of Anisakis larvae parasitic on Pneumatophorus japonicus for different weights of fish body. • 1. - Weight (g). 2. Number examined. 3. Number positive (%). 4. Number of larvae detected. 5.. Minimum. 6. Maximum. 7. Average. 8. Total.

IV) Parasitic conditions of the larvae for different lengths of fish body

Okumura.(1967) investigated the relation between the fish body length and the nueer of parasitic Anisakis larvae with Pneumatophorus japonicus, Trachurus japonicus and Dentex tumifrons (Table 11-21). Asa result, the number of parasites increased with increasing fish body length

almost identically in all three fiSh species. The number .,-- • of parasites increased rapidly in Pneumatophorus japonicus • of body length more than 35 or 36cm, in Trachurus japonicus

of body length more than 33 or 34cm, and in Dentex tumifrons of body length more than 23 or 24cm, and a large difference was noticed compared with those of shorter body length. Okumura (1967) confirmed that there is a definite relation between fish body length and the number of parasitic larvae, and he presumed that the cause lies in the different times of contacting pollut'ed water areas.

c1 ;j ® :••• .7 •', "7 7 ':•••• © 3* 1

7 1 fi, cpee • (?..) eb I ,lt•-) i* 1 à ..t. ,„ ..es ,.., s":-.,' (cm) ''' j ft, -. ,à .4. 5, ...,n ,i, , ..", 1 ftC2i '`* A .k.'":, r) c..) ;j: `'-' '` • • r. • ..J c., ,-)1- Le) ü' 1.. S .) -•t. ;• )r..)' g .e. '''''' `. 1'::.' el) 0 f"." " 17-18 0 Ci. 20 111 5.6 0 24 19-20 15 88 5.6 0 .23 21-22 17 42 1.3 0 5 23-24 4 3 0.8 0 3 10 20 2.0 0 5 8 47 5.9 0 42 25-26 8 14 1.8 0 8 7 3 0.4 0 3 13 326 25.8 0 64 27-28 12 173 12.4 0 48 11 1.8 0 10 29-30 17 238 14.0 0 115 31-32 21 77 3.7 0 9 5 . 106 21.2 0 55 33-34 46 279 6.1 0 26 11 1,570 142.7 0 404 35-36 33 382 11.9 6 20 5 892 178.4 14 436 • 37-39 11 189 17.2 1 47 40-43 19 422 22.2 2 109

Table 11-21. Fish body length and the number of larvae detected. 1. Fish body length. 2. 3. rachurus japonicus 4. Dentex tumifrons 5. Number of fish examined. 6. Number of larvae detected. 7. Mean number of larvae per fish. 8. Number of larvae detected in one fish. 9. Minimum. 10. Maximum. 72

(4) Monthly or Seasonal Distribution of Anisakis Larvae in Pisces

It is considered very important in preventing infection to know how the number of parasitic larvae in a fish species varies during the year. There are reports by Okumura (1967), Koyanagi (1967) and Kato et al. (1968) concerning this point. Although for various reasons none of these investigations were conducted throughout the year, and the number of fish examined varies in each month, they are summarized below. According to the monthly detection of Anisakis larvae parasitic on Pneumatophorus japonicUs from 1964 to 1967 conducted by Koyanagi (1967, Table 11-22), their numbers were high in.May, June, July and August in any year. Next, results of the monthly rise and fall of the number of parasitic Anisakis larvae in pneumatuhoEm japonicus and Trachurus japonicus investigated by Okumura are demonstrated in Table 11-23. Looking at the 'results, tendencies can be noticed for the number of parasites in Pneumatophorus japonicus to increase in the spring and summer months of April, June and July, and for those in Trachurus japonicus to increase in the winter months of December, January. and February. This conclusion would have been-more obvious if the fish body weight, length and age 73

had been recorded. On the other hand, Kato et al. (1968) conducted an investigation into the seasonal changes in detection level of Anisakis type T and II larvae in the three species Trachurus japonicus, Scomber japonicus and Ommastrephes sloani Eacificus (Table II-24). According to this, the detection level was Comparatively high from February to March in Trachurus japonicus, and from December to March in both Scomber japonicus and Ommastrephes sloani pacificus. Even though a variation in monthly, seasonal detection level of thebe larvae was noticed from the above three investigation results, we should not consider this a definite conclusion, because of the danger from a food hygienic standpoint that we might give an impression to consumers that the degree of infection by theselarvae varies through the year, there being a season when theY do not have to worry about Anisakis disease when eating raw fish.

(5) Distribution of the Larvae in Pisces and Cephalopoda with Different Habitats

Although the relation between various fish species ' and the parasitic conditions of the larvae has already been described in Section (1), the relation between the different

R (%) e e 14 , CD 8 5je

; 249 227 ( 91.2 ) 1.578 0 28 7.0 9.2 8.7 2 214 198 ( 92.5 ) 1,324 0 27 6.7

3 101 101 (100.0 ) 946 1 28 9.4 8.3

4 22 20 ( 91.0 ) 86 0 12 3.9 8.9'

E. 64 56 ( 87.5 ) 702 0 80 11.0 f6.1 10.0 16.7 ' 6 130 117 ( ) 1,315 0 72 7 101 79 ( 18.2 1,055 0 72 16.0 14.8

8 32 31 ! 96.9 ) 326 0 38 10.2 13.5 9 • 3 1 ( 33.3 9 0 9 3.0 5.9 9.4 12.7 10 167 164 1 97.6 ) 1.563 0 40 11 77 69 ( 29.6 701 0 51 8.9 12.2

12 142 139 ! 9/ .9 ) 1,231 0 43 8.3 12.8 0) 11. 1,362 1,262 ( 92.3 ) 10. 184 0 80 8.3 14.1

Table 11-22. Èesults of monthly detection of Anisakis larvae parasitic on Pneumatophorus japonicus (Mar. 1964 - Feb. 1967) 1. Month. 2. Number examined. 3. Number positive. 4. Number of larvae detected. 5. Minimum. 6. Maximum. 7. Average. 8. Number of larvae per one kilogram fish body. 9. Total.

(:D 0-, 7 Ji 1 0! :fi af, 1 11C4- Q fhrz r (r)se ell: VS: e)5.1;fi 1. Month. 5 12 2.4 7 1,568 224.0 2. Pneumatophorus japonicus 2 44 286 6.5 3 440 146.6 3. Number.of fish examined. 3 21 108 5.1 11 75 6.8 4. Number of larvae 16 224 14.0 6 13 2.1 detected. 5 5. Number of larVae per 6 16 272 17.0 fish. 7. 25 256 10.2 Trachurus japonicus 8 9 73 8.1 9 22 136 6.1 32 349 10.9 - 10 10 60' 6.0 -- 11 8 57 7.1 2 94 47.5 12 20 137 6.8 4 454 113.5

Table 11-23. Number of larvae detected for different months.

b il li v 0 7 .i'..*- v -€) 1- ,.: .. it- ri, 4 '7 Ee Q) sf- fi efee (re V4-, % tecifirlt ,p,.e 11±T%4 Ve.tfi er,,t VV.96 ge vae 42.7 8 31 0 0 13 11 85 14 3 21

10 - - 8 50 3 • 0 0

12 - - - 15 12 80 41 17 42 43.1 23 1 4 42 40 90 21 9 43 2 34 .13 38 11 11 103 22 15 68 3 107 22 21 so 48 96 3 1 33 4 56 0. 0 - - - - - % It 275 37 13 142 127 87 Ill 47 42

Table II-24. Seasonal infection percentage of genus Anisakis larvae. 1. Date in Showa years. .(Showa 42-= 1967, Showa 43 = 1968) 2. Species. 3. Trachurus japonicus 4. ScoMber japonicus 5. Ommastrephes sloani pacificus 6. Number examined. • 7. Number infected. 8. Infection percentage. 9. Total.

habitats of the fish species and the Anisakis larvae infecting conditions isadded here. There are reports (Table 1I-25) by Kobayashi and Kagei et al. (1968) and investigation results (Table II-26) by Kato et al. (1968) concerning this. From Table 1 1-25, it can be seen that the percentage of fish parasitized by this larvae is 'high fn such fish as Theragra chalcogramma, Oncorhynchus masou, Clupea pallasi, • Scomber japonicus and Ommastrephes •sloanf pacificus, etc. 76

distributed mainly in the waters around northern Japan and in fish distributed in the coastal waters of the Pacific Ocean and the Japan Sea which migrate north and south seasonally, and that generally the infection does not occur as much in-those fish which are sedentary or inhabit warm waters.

Table 11-26 shows almost the same results as • Table 11-25... The degree of parasitism of Anisakis type larvae is high in those fish which migrate north and south seasonally such as Ommastrephes sloani pacfficus or Sardinops melanosticta and those which inhabit northern seas such as Clupea pallasi and Theragra chalcogramma, and the detection percentage of type II larvae is high mainly'in large fish such as Katsuwonus pelamis and sharks which inhabit southern 32 offshore waters.

çb . Ill Ci rsA e*n .'1.- 71,1 ft)ez ®(_DnruM.Z., it (5 It

0,s?) ( A ) :.t.. a .e•,-,%ie 1- 4...c. A 'r F ,) 7 5 119 100 % 43 . 8 80% 4 . 5 ;I * rt " ) L -iee-r 5 ''t e. 5 v.7.0 10 100 20.0 40 39.0 . c9 • -7 ",' 0 48 96 32.6 25 1.0 " .'....., is 100 77 4.6 2 ..0 uPtIt..q. 9101 0 ( 8 ) a *;e1, c•J:,e. ,7 285 51 31.1 0.4 1.0 iI 5 ,5;4) ,,_,) L. r. '''! , e (3) 573 81 9.4 62 4.2 it 1 81'I: iti-.. MT ''...' 4 1, 0 755 42 1.7 19 1.5 911 D. -r61, 0 . ,...... , e 20 5 1.0 0 0 UM'. ti 7. 5 '.., e 1. -.... 20 0 0 0 . 0 q 811 -7 5 •., 2 F :..., 2 100 li( 76.5* 100 0 ? ? ( C ) le,lie.1.3.r.. a ( e 7' i, 7 "". '1` '' (b L .. chÉ lie) ,,,.., h -.% >1- () 10 90 6.1 0 0 fiv*It • (£) ivI,' 0 20 . 55 2.5 15 1.7 fi. ( •0) • " ii -Y 4 (2) 12 33 1.3 0 0 . 0 . 0 0 Ifi fell 1® , v 0 0 20 0 ( 0) FP! ‘ri,lb -c-1 Lee; 4 .> if .7 419 13 0 0 0 0 1: 11 esVO • 71. 4111 G9 t.cr, 7 =et, 4C) 10 0 0 0 0 rtuir..mes (D 7 t 4 h 0 23 0 0 0 0 0 0 0 0 7 20 0 0 • 0 0 7 t. 9 4,7 24. 0 0 . 0 0 CEi) e fi

Table 11-25. Parasitic conditions of Anisakis larvae in Pisces and Decapoda, and their evaluation as a source of infection towards humans.

1. Distribution. (A) Distributed in the waters around northern Japan. (B) Distributed in the coastal waters (Pàcific, Japan Sea) 'of Japan and migrating north and south. (C) Distributed over a wide area in warm waters and having wide migration habits. (D) Distributed in Japanese coastal waters and which do not migrate much. 2. Species. 3. Number examined. 4. Percentage parasitized. 5. Mean number of larvae. 6. Parasitism inside the musculature. 7. Percentage parasitized. 8. Mean number of larvae. 9. Food habit. 10. Theragra chalcogramma 11. Oncorhynchus masou 12. Gadus macrocephalus 13. Clupea pallasi 14. Trachurus japonicus 15. Pneumatophorus japonicus

16. Ommastrephes sloani pacificus 17. Cololabis saira 18. Sardinops melanosticta 19. Lampris regius 20. Katsuwonus pelamis 21. ..I.D_neulatohorta,eiriocus 22. Sarda orientalis 23. Thunnus thynnus 24. Oplegnathus punctatus 25. Limanda yokohamae 26. Decapoda sp. 27.Sepia sp. . 28. Sepia esculenta 29. Doryteuthis bleekeri 30. Po1yphagia. 31. Crustaceans, fish. 32. Piscivorous. 33. Zooplanktons. 34. Phytoplànktons. 35. Sessile food. 36. Benthos.. 37. Mostly Anisakis type II larvae.

0 CD CD Anisakise)‘ 1 M Anisakisd V Raphidescaris Contracaccum Z5)- . Pie,It 411114.* jgd 1 ire - 1,4z 4e, ,I I eft z% k* dip Pe czy I. Qe I* ci) 28 286 15 67 4 . 5 6 14 6 42 (53.5) (23.4) (14.2) ( 1.7) (21.4) ( 4.8) (21.4) (14.6)

10 211 3 20 6 114 0 0 ID 14 ' 930 (71.4) (22.6) (21.4) ( 2.0) (42.8) (12.2)

7 114 1 4 1 3 4 14 11 262 (63.6) (43.5) ( 9.0) ( 1.5) ( 9.1) ( 1.1) (36.3) ( 5.a) 10 18 9 13 0 0 .2 4 qp 17 62 ( 58. 8 ) (34. 4 ) (52.9) (20.9) (11.7) ( 6.4) 42 410 17 42 13 131 12 60 D It 70 1.540 (60.0) (26.6) (24.2) ( 2.7) (18.5) ( 8.5) (1 8 . 5 ) ( 3 . 8 )

1;1.)(1)11a*iàkiii.:5PL. iteEe);£1 -r t, (. 0. .z ve4. V 7 7> m •)) 01113*b1.153 1%1A5T. LL. ELM 1- 51,69 euitz,,etJ.: elt t F /7'3. --ey3 7 E) EDet,,e. (Itzffie,eu) to)(n,**7e 7 eo, r,:;*(veotzE)

Table II-26. Parasitic conditions of the subfamily Anisakinae nematodes for different fish inhabiting waters.

1. Classification. 2. Number of fish species. 3. Number of fish. 79

4: Anisakis type I

5. Anisakis •type II 6. Number of fish species. 7. Number of fish. 8. Total. . 9. (Note):

. Fish species which are dietributed in the

- - . coastal waters of Japan . and are relatively sèdentary . (Konosirus punctattis, Lateolabrax japonicus, Pagrus major,.Fugu yermiculare porphyreum, LoPhius litulon, etc.). Fish which are distributed in the coastal waters of Japan and migrate north and south (Sardinops melanosticta, Cololabis saira, Ommastrephes sloani pacificus e etc.). Fish which inhabit northern waters (Clupea pallasi e Theragra chalcogramma, Pleurogrammus azonus, Gadus macrocephalus, etc.). Fish which live or migrate in offshore (mainly southern) wate:cs (Katsuwonus Pelamis, Neothunnus albacôra, Rhincodon typus, Makaira Mitsukurii, etc.).

Summary

In the above, we ecologically sumMarized the nematodes of the subfamily Anisakinae (genus Anisakis) as pathogenic organs of anisakiasis by division into . the five items of fish speciep, marine mammals, fish bodies, Months or seasons, and habitats.. As a result, we see that the:ecology of the subfamily Anisàkinae (genus Anisakis) nemàtodes (mainly Anisakis larvae) exhibits various conditions. It is very significant for grasping the degree of 80

pervasion of the natural world by these nematodes and moreover for investigating the source and route of anisakiasis infection, to understand the life history and ecology of the genus Anisakis nematodes. It is obvious from the ecological_observation results above that we are always exposed to the danger of anisakiasis infection by the consumption of raw fish. From a standpoint of food hygiene, it is necessary to understand first of all the pathogenic oTgans, examine the conditions of pollution by the pathogens and assume the source and route of infection, and on the basis of these we must isolate the infection factors of anisakiasis and devise countermeasures for preventing infection towards humans. From this standpoint, a major prerequisite for the problem of preventing anisakiasis is the establishing of the life history and ecology of the genus Anisakis nematodes which are the pathogenic organs of the disease. Needless to say the detection of Anisakis larvae and pathogenesis of anisakiasis are not necessarily related to each other, but it will be true that their detection is , one of the larger factors. The primary problem assigned to us who are studying 34 food hygienics is to differentiate the nematodes of the genus Anisakis and to understand them ecologically. In any case, the research of the life history and ecology of these 81

nematodes is qssential and indispensable for the health of our nation's population, in view of dietary habits in our country. 82

III. ANISAKIASIS

Larvae of the genus Anisakis are parasitic in marine fish, especially in the musculature of Theragrachalcogramma, Sepia esculenta, Scomber japonicus, PleurouLamus azonus, etc. as mentioned earlier. When meat of these fish is consumed raw as sashimi (sliced raw fish) or sushi (raw fish dish), Anisakis larvae may also enter the human alimentary canal with the fish. It is known that in such cases, the larvae invade the stomach or intestinal canal wall and cause diseases such as eosinophilic gastric granulomas or acute circumscribed enteritis, although they do not live for a long time. These diseases are called gastric or intestinal anisakiasis. In the former chapters, we have summarized those research results to date mainly concerned with the classification and morphology of the Anisakis-type larva (Anisakis- like larva) in particular, which is considered to be the causative agent of thii disease. . In this chapter, we are going to deal with the general idea of the disease called anisakiasis which is caused by these larvae and at the saine the facts to some extent. time we will endeavour to clarify The historical background of anisakiasis research to date, . .its epidemiology, clinic and pathology, diagnosis and therapy will be summarized item by item below. 83

Historical Background of Anisakiasis Research

Inflammatory diseases which cause the infiltration of eosinophilic'leucocytes in the human body can often be seen in the_bone, subcutaneous cell tissues and other parts, and many examples of such diseases in the digestive system have been reported for many years, as eosinophilic granulomas of the stomach or in the intestinal wall or as acute localized enteritis. Antigen-antibody reactions and unsaturated fatty acid aldehydes mainly from ascarids, etc. have been known as the cause of these syndromes, but digestive diseases which cause inflammation with eosinophilic infiltration have been vaguely considered as allergic disorders and the real cause has not been known. However, our predecessors have often pointed out the possibility that parasites such as ascarids could have entered and caused these syndromes. In these cases the local lesion was recorded to have the characteristics of an eosinophilic phlegmon or a helminthic abscess, and in our country there are reports on this by Shioda (1940) and Sunabara (1954), etc.. The casuistics of eosinophilic granulomatosis in the stomach are not new, and according to Feyster (1953), the first record was by Jaeger (1932),-and .Vanek (1949) described six examples,of this symptom found in the pars pylorica and by attributing the cause to an allergy he considered that even the granuloma state is not abnormal. 84

In our ciCiintry they were also all reported as cases of eosinophilic granulomas in the stomach wall by Koshiie (1954), Motojima (1955), Ishii et al. (1956), Sano et al. (1961), Uchiyama et al. (1961) and Tsuchiya et al. (1963), and it was recorded that sections of larval nematodes were observed in some of the resected tissue specimens. • 'Apart from the reports of these syndromes, Hitchcock . 36 (1950 discovered ascarid-like larvae in the excrement of about 10% of an Eskimo population,' and Buckley (1951) observed one helminth expplled from a human oral cavity and identified the parasite as the genus Porrocaecum or the genus Anisakis, but the pathogenicity to humans was unknown. Beaver (1956) introduced the concept that when the parasites(ova) of other animals are consumed by man, they can enter and migrate through the alimentary Canal wall - visceral larva migrans. Subsequently, Van Thiel (1960) observed cases in which the same nematode larvae as Hitchcock recorded were thought to have been present or to have penetrated in eleven examples of acute enteritis in Dutch people who ate raw herrings and he clarified that all of these were eosinophilic granulomas. At first he presumed.these larval nematodes to be Eustoma rotundatum, but later he,corrected this and identified them as Anisakis and proposed that all the cases should be called 'anisakiasis. Stimulated by these human casuistics, attention has at last been brought to the pathogenic features, 85

morphology and •the life history of these nematodes. Myers (1960) in Canada established the pathogenicity ofthese larvae by animal experiments, and Kuiper et al. (1960) reported three interesting cases which were thought to be caused by Eustoma rotundatum and in one of these cases hé observed a larval nematode which had penetrated into the wall of the small intestine. Recently,*Ashby et al. (1964) undertook a clinical, pathological analysis of the 89 cases of eosinophilic granuloma in the human alimentary tract thought to have been caused by E. rotundatum during the previous 25 years, and pointed out that a massive eosinophilic infiltration was observed in each case. More recently, Merkelback (1965) of Holland observed that one example of localized eosinophilic granuloma found in the submucosa of the rectum showed a positive reaction in a complement fixation test with the antigen of Anisakis and he attributed the pathogenicity to the migration of this larva . In contrast to the research in other countries, our research has only been published intensively in the last few years. Otsuru et al. (1957) reported two cases in the past of localized enteritis caused by intestinal wall migration of ascarid-like larval nematodes and considered them to be young larvae of the genus Ascaris at that time, but

. later reconsidering on the basis of Beaver's (1956) theory of visceral larva migrans, he corrected this and regarded 86

the larvae as Anisakis-type. Since then, from around 1963, many clinical cases have been reported ail over the country (Nishimura, 1963; Kunishige, 1964; Okamura, 1964; Oshima, 1964; Asami, 1964,1965; Yamaguchi et al., 1964,1967; Otsuru, 1964,1965,1965; Yoshimura and Yokokawa, 1964,1966; Kikuchi et al., 1966; rshikura,1965,1966,1967,1968; Ishikura and Kikuchi,.1967). In particular, Asami et al. (1964) sent pathological resected specimens from their patients to Beaver for the first. time from Japan, and they clarified that it was the saine species. as the one Van Thiel reported and identified, and reported that most of the cases in the past were caused by nematode larvae of the genus Anisakis whose hosts are marine mammals. The eosinophilic granuloma caused by the migration of Anisakis-like larvae in the alimentary canal wall has aroused much attention as a new problem both here and abroad not only in the parasitological field but also in the clinical field, especially from the standpoint of digestive diseases. As a result, it was realized that anisakiasis infection possibly would have been included to a high degree in cases which had been wrongly diagnosed as acute Crohn's disease, acute appendicitis, ileus, chole- lithiasis and cholecystitis, etc. because anisakiasis shows 'symptoms similar to those of stomach cancer, peptic, and duodenal ulcers, etc.. In 1965 then, the Ministry of 87

Education General Research Group (group head: Masamitsu Otsuru, full professor of Niigata University) was organized and gradually new facts on this disease have been found. In the abeve we have reviewed the historical process up to the time when the general idea or the . actual faet of anisakiasis was grasped from the research here and abroad, and we think it proper to consider that the history of serious research on this disease began after 1960 approximately.

Epidemiology of Anisakiasis

It was found from medical samples (pathological tissue resection specimens) or data that many cases of eosinophilic granuloma considered to be caused by the migration of Anisakis-like larvae into human bodies had been included in the former reports of phlegmones, abscesses and granulomas in which eosinophilic infiltration focussing on the stomach or intestinal wall was very marked, and the re-investigation of these or similar diseases became necessary, and at the same time a new epidemiological study of helminthic granulomatosis was necessitated. Some clinical cases of this disease had been reported

by the above-mentioned researchers in our country before 1966, 88

and it was Yoshimura (1966) who investigated them collectively from the standpoint of epidemiology. Subsequently, Ishikura (1968) carried out epidemiological investigations of acute localized enteritis, i.e. intestinal anisakiasis, in Hokkaido (especially_the area around Iwanai Town) and Otsuru (1968) reported 53 cases of phlegmon, abscess or granuloma which broke out in the area:of Niigata Prefecture after 1955. There is also a review paper by Ishikura (1968) on the condition of anisakiasis outbreaks in Japan. Referring to these, the present state of epidemiological research on anisakiasis is described below.

(1) Geographical Conditions of Outbreaks

There is a report by Yoshimura (1966) concerning the number of outbreaks of phlegmones, abscesses and granulomas' due to the migration of Anisakis-like larvae classified according to the prefectures in our country (Table III-1, Fig. III-l). As is obvious from the report, these cases occur all over the country. It appears that there are some regional differences in the number of outbreaks, but .this is not clear. Is.an exception, the number of outbreaks in Niigata Prefecture is large, perhaps because Otsuru et al. extensively examined 89

Table III-1- Prefectural total of Anisakis-like larva migrans in human alimentary canal. .(as of 1966)

Name of prefecture Number of Source cases

Hokkaido 6 Kikuchi, Imamura, Asami . Akita 1 Otsuru . Fukushima 7 Otsuru, Kojima Niigata . 27 Otsuru, Kojima Toyama . 3 Otsuru, Yoshimura Ishikawa 5 Yokokawa - Yoshimura Ibaragi . 2 Asami, Yoshimura Chiba 5 Asami, Yoshimura Tokyo 3 Asami, Yokokawa - Yoshimura, Motojima Saitama . 1 • Asami Shizuoka 1 Yoshimura et al. Gifu. 3 Oshima - Kagei, • Morishita - Ojima Aichi • 1 Morishita - Ojima Mie 1 Morishita - Ojima Osaka 2 Nishimura Hiroshima 1 Inatomi Yamaguchi 3 Hosokawa Kagawa 1 _ Yamaguchi Tokushima 3 Yamaguchi : Ehime 1 Yamaguchi Fukuoka 1 Oshima - Kagei Kumamoto 3 Okamura Kagoshima .3 Oshima - Kagei, Hashiguchi Unknown 5 Otsuru, Yamaguchi, . Yoshimura

(D it'M in 6

tkEE 1

th427

G etil 3 Ute /11 5 e.k-142(D. (i) ria% itt3i 5 ® OthrJ3 s . ,g 1@so ig,g1 1 •+0

L. Nk* 3 A,

n't.:2e, 3 ,74.■ I I •! V9 .•I u2-"/ • Fig. III-1. Total per prefecture out of 84 reported cases of eosinophilic granuloma in the human alimentary canal (caused mainly by the migration of Anisakis-like larvae). As of 1966. 1. Hokkaido - 6 2. Akita - 2 3. Niigata - 27 4. Toyama - 3 5. Ishikawa - 5 6. Hiroshima - 1 7. Yamaguchi - 3 8. Fukuoka 1 9. Kumamoto - 3 10. Kagoshima - 3 11. Ehime - 1 12. Kagawa - 1 13. Tokushima - 3 14. Osaka - 2 15. Mie - 1 16. Aichi - 1 17. Gifu -3 18. Shizuoka - 1 19. Tokyo - 3 20. Chiba - 5 21. Saitama - 1 22. Ibaragi - 2 • 23. Fukushima - 7

the patients in,that area. The results by Ishikura et al. (1968) are not included in Yoshimura's report, but will be described in Section (4). Ishikura et al. (1968) published a report on 191 cases of acute localized enteritis occurring in Hokkaido,during the year 1965 (Fig. III-2).

et F. 513) (I) 0

0 1 0 o Ce ffigiill Ott 2(1) 0 ° 21 6 ‘,. 74rie3g) efflD.1 , 116M; 6121 (e) kennel) 04 4 9 4 ÇDMM12./ 1 (15 ;11t 6(ii) 6.-, (D”M 3 ° C ° 11e50Œ-5 0 i /A4.11m u9 *den ) 0 iilhe..5 We) 3 ° 5 _ero

tItc 18 /1 (71C)

(i.1) 1965: 191 /3 ( 7' = 4-1* ), 1.e5È37-el) 151] 0, 19604- : uof351

Fig. 111-2. Distribution of outbreaks of localized enteritis in Hokkaido. 1. Wakkanai 5(3) 12. Iwanai 4/1(8) 2. Rumoi 11 13. Sapporo 50(25) 3. Monbetsu 1 14. Obihiro 12(1) 4. Otaru 6(2) 15. Shibecha 3 5. Asahikawa 3(7) 16. Kushiro 10(3) 6. Engaru 6 17. Nemuro (1) 7. Abashiri 2(1) 18. Oshamanbe 5 8. Rausu 3 19. Tomakomai 5 9. Sorachi 12/1(11) 20. Muroran 10(1) 10. Kitami 4 21. Urakawa 5(1) 11. Shibetsu 6 22. Hakodate 18/1(7) 23. 1965: 191 / 3 ca ses (in which Anisakis larvae were foun707----- 24. 1960: (76) cases 92

Concerning the «distribution of outbreaks by area in Hokkaido, 39 56 cases were in the Okhotsk Sea, Japan Sea and Tsugaru Strait side, 35 cases were on the Pacifie Ocean side, and 100 cases were in the interior. Although Sapporo is an exception because of a large population, it is presumed that one of the reasons why so many >cases were found in the interior • such, as in Obihiro, Asahikawa and Sorachi, etc. is that the • larvae have moved from the visceral organs to the muscular parts of the fish because there is a long time between capture and consumption.

(2) Dependence of Outbreaks on Sex

According to Yoshimura (1966, Fig. III-3), the number of occurrences in men was 62 cases, that for women 24 cases, that is, 2.5 times as many in men as in women. Also in the report by Otsuru (1968) 36 out of 53 cases were in men and 16 cases were in women. In the report by Ishikura et al. (1968), the ratio between men and women was 115 : 76, 1.5 times as many in men as in women. 93

(5) (±) yeî-- il tJ

*•■•.-1

—M M 40 50 gl, Fig. 11I-3. Distribution of nosogenesis by age and sex. 1. Number of cases. 2. People. 3. Men. 4. Women. 5. Age.

(3) Dependence of Outbreaks on Age

According to Yoshimura (1966, Fig. III-3), the disease occurs mostly between the ages of 30 and 50 both in men and women, and half of the total number of outbreaks are in this age group. Otsuru (1968) compared the two types of .gastric type and intestinal type and reported that the gastric type occurs between the ages of 20 and 59, particu-

larly between the ages of 40 and 49, and the intestinal 94

type occurs in the age range of 10 to 69. If it is generally like this, there cannot be said to be any relation with age. On the other hand, Ishikura et al. (1968) reported on the dependence of outbreaks of intestinal anisakiasis on age (Fig. III-4) that many occur between the ageeof 11 and 40, and the morbidity rate is high between the ages of 11 and 30 especially.

0-10 (10) ® 1. 11 20 is€ (51) 21-30 (67) 31 40 (37) 41-50 (12) 51 60 (10) (D — (4 )

I I 4 t 10 20 30 40 50 60 70 ez 0

Fig. III-4. Dependence of outbreaks of localized enteritis › on age. Ï. Number of occurrences. 2. Age classification. .3. Years.

(4) Conditions of Distribution of Gastric Type and Intestinal Type

When Ishikura (1968, 1969) compiled the already 'published reports and some questionnaires which were brought

in from 53 institutions across the nation, there were 278 .95

cases of anisakiasis which had occurred by the end of 1967. Among them, 196 cases were gastric anisakiasis, 77 cases intestinal anisakiasis, 5 cases other. When these results were classified into gastric and intestinal types and the conditions of the outbreaks were examined, 1y dividing the country into nine blocks, it was found that the gastric type occurs more in the south, and the intestinal type increases towards the north. For example, in Kyushu there were 28 gastric type cases and 2 intestinal cases, whereas in Hokkaido there Were 9 gastric type cases, 32 intestinal type cases (Fig. III-5). Although this is an interesting observation, the reason. is not yet known. Moreover, Ishikura (1969) studied the dependence of

the outbreaks of 172 cases of gastric anisakiasis and 77 cases of intestinal anisakiasis on sex and age (Fig. III-6), with the results that the former disease occurred in 117 men compared to 55 women (2.1 : 1.0) with many occurrences between the ages of 30 and 59. In the latter disease, the ratio of the outbreaks between men and women was 1.8 : 1.0, being also more in men, and many occur between the ages of 10 and 39, this being a younger group compared with that of gastric anisakiasis. We continue to introduce Ishikura's report (The Hokkaido News, 1968). Since a disease called 'anisakiasis' 11› was established in 1964, there are 278 cases reported up to 96 ,

Fig. 111-5. Variations in outbreaks of gastric and intestinal anisakiasis in different areas. 1. Gastric anisakiasis. 2. Intestinal anisakiasis. 3. Kyushu. 4. Shikoku. 5. Chugoku. 6. Kinki. 7. Chubu. 8. Kanto. • 9. Niigata. 10. Tohoku. 11. Hokkaido. e

now, these being only those in which larval bodies were found in the affected parts. Among these, 52 cases occurred in Hokkaido. There were 122 cases considered as undoubtedly anisakiasis from the histological examination of the lesion alone in Hokkaido, and if similar ones, although their histological examinations are not complete, were included, the number of cases would exceed 700. There are 500 cases in the country which were confirmed to be anisakiasis by the examination of pathological 97

and histological samples (The Science News, 1968). Ishikura et al. (1967) found that the 129 cases of intestinal anisakiasis which occurred in Iwanai Town, Hokkaido, between 1955 and 1967 occurred at the same time as the fishing season of Theragra chalcogramma ancLi'leurommillu_s_ azonus, etc. (Fig. III-7). It was presumed from this result that the outbreaks of this disease in the Iwanai area were caused by the consumption of these fish in the raw state.

Fig. III-6. Dependence of outbreaks of anisakiasis on age. 1. Number of cases. 2. Age. 3. Years. 4. Gastric anisakiasis. 5. Intestinal anisakiasis. 6. Men. 7. Women. 98

ICIY so

40 1 019601If, ( ',If-)

20 .1

19654t.it ( 1 ,r )

/ • I • é 4 1 / 1 2 34 56 7 8 91011 121'3 C) r 4 i) •Y v 3" 3

qà).71 1=1 (D4t (ilt S ."

Fig. 111-7. Monthly outbreaks of localized enteritis and the catch of marine fish. 1. Number of outbreaks. 2. 1960 total (five years). 3. 1965 total (one year). 4. Month. 5. Theragra chalcogramma 6. Sepia esculenta 7. Pleurogrammus azonus 8. Gadus macrocephalus 9. Scomber japonicus 10. Clupea pallasi 11. Cololabis saira 12. Paralichthys olivaceus 99

In particular, according to Ishikura et al. (1967), there is a tendencifor the morbidity rate of anisakiasis to be high in the northern part of our country, that is, Hokkaido, Tohoku and Hokuriku districts. The reason was presumed to_loe because the marine mammals which are the final hosts of Anisakis adults exist in great numbers in the waters of northern Japan and therefore, the parasitid percentage of Anisakis larvae is high in fish of the northern Japan seas.

(5) Summary of Epidemiology

Today it is considered that there is a very significant relation between the helminthic granulomatosis or phlegmon › in the alimentary canal and the parasitism of Anisakis larvae,or they have some connection with each other in some • 42 form. It is thought that the establishment by Otsuru et al. (1957) of the idea of visceral larva migrans gave important encouragement for solving this new type of parasitic disease of anisakiasis which has been occurring a great deal in our country. The thorough investigation of the number of out- 'breaks or other epidemiological studies of this disease are essential now and must be continued in the future. It 100

appears that there have been quite a few cases which were treated latently without being officially published or which were given an incorrect preoperative diagnosis.

Clinic and Patholoey Of Anisakiasis

In our country gastric type anisakiasis accounts for two-thirds of all the cases, which is different from the situation in Holland where there are more intestinal type cases (Van Thiel, 1960, 1962). In Honshu, Shikoku and Kyushu districts, the cases are generally gastric type and in the Hirosaki area gastric and intestinal type cases are approximately equal in number, and in Hokkaido most are cases of acute localized enteritis developing in the small intestines. As there are many intestinal type occurrences in Hokkaido where the morbidity rate is high, it means that there is more intestinal type disease in our country as a whole. On the basis of clinical, pathologic, histological states, there are considerable differences in the reaction behaviour of the host between the two types of gastric and intestinal anisakiasis. Now, we woUld like to describe the 'clinical and pathological aspects of these two types,

* Translator's footnote: This appears to be a contradiction in the original'. 101

referring mainly to the clinical, pathologic histological state casuistics of the gastric type by Yoshimura (1967) and by Otsuru et al. (1968), and the reports on intestinal aniSakiasis by Ishikura et al. (1966, 1967, 1969).

Clinic

The diagnosis of anisakiasis has not been given until after the condition was diagnosed erroneously,as stomach cancer, peptic ulcer, stomach tumor or chole-lithiasis, etc. in the gastric type; or as acute appendicitis or ileus, etc. in the intestinal type, in preoperative diagnosis and then rigorous examination of patients carried out following laparotomy. 'These incorrect preoperative diagnoses happen because there is much difficulty in the clinical classification of this disease among other diseases such as those above. - If we consider this in another way, it can be said that this disease is a helminthiasis Which acts peculiarly towards the stomach or intestine. From the preoperative diagnoses of 92 cases in the report by Yoshimura (1967, 1968, Table III-2), the 53 cases of Otsuru (1968, Table III-3)

and the gastric and intestinal anisakiasis cases of Ishikura 43 •(1969, Table III-4,5), it will be realized that for both gastric and intestinal anisakiasis the preoperative diagnosis 102

is very difficult and many cases of gastric anisakiasis are erroneously diagnosed as stomach cancer, stomach tumor or peptic ulcer, and intestinal anisakiasis as acute appendicitis or acute localized enteritis. According to Otsuru (1968), the period during which complaints of discomfort are received varies in the gastric type, the shortest being about one week, and the longest • being two years, and in the intestinal type, considering periods of one day, one to three days and three to seven days, 25% of the cases are acute ones of within one day, being the largest group, the next largest group are those between one and three days and the third are between three and seven days, the period being less than one month for 65% of all the cases. There was no definite tendency in the period between disease occurrence and operation in the 92 cases of Yoshimura (1967, 1968, Table III-6). According to the reports by IShikura (1968, 1969) - and Otsuru (1968), the main characteristics of the subjective symptoms are precordial pain, a feeling of plenitude in the precordial region, vomiturition, vomiting, and eosinophilia in the peripheral blood in the gastric type, but sometimes there are no symptoms. In the intestinal type the characteristics are abdominal colics, vomiturition, vomiting,

Symtoms of irritated peritonaeum and eosinophilic leucocytosis 44 (Fig. III-8). Ishikura (1967) presented the clinical 103

symptoms of 13 cases of intestinal anisakiasis and in 1969 he made a comparison of the clinical symptoms of acute appendicitis and acute ileus which are difficult to distinguish from intestinal anisakiasis in clinical diagnosis (Table III-7). According to the reports by Yoshimura (1966, Table III- 8) and Otsuru (1968, Table III-9) concerning the lesional parts of this disease; the corpus ventriculi and vestibulum ventriculi are the. parts affected in the gastric type, but the pars pylorica cannot be omitted. In the intestinal type, the whole area of the ileum including the distal part is the main affected part. When the macroscopic changes of the lesion in the gastric and intestinal types are compared, they are generally common to both types, a localized phyma buried in the'submucosa of size between a small finger tip and thumb tip being formed in both types. Some were observed to. have small ulcers, fovea and small haemorrhagic erosions near the phyma buried in the submucosa (Yoshimura, 1967). On the other hand, the macroscopic features observed at the time of laparotomy in intestinal anisakiasis cases are shown in Table III-10. In these cases haemorrhages and opacification in the intestinal mucosa, edematization in the intestinal canal, *abdominal dropsy, etc. were common features. 104

Table 111-2. Preoperative clinical diagnosis items. stomach cancer or stomach tumor 34 cases (37%) peptic ulcer or duodenal ulcer 18 (19%) acute appendicitis or acute abdomen 15 ( 1 7%) ileus,intussusception 3 chole-lithiasis, cholecystitis 2 terminal ileitis anemia 2 tuberculous peritonitis 1 pancreas cancer 1 diverticulitis 1 unknown 13 (15%)

Total 92 cases

Table 111-3. 53 cases (clinical diagnosis) in Niigata and neighbouring prefectures.

Cases in stomach Cases in intestine stomach cancer 8 appendicitis 5 stomach tumor, polyp 8 ileus 3 peptic ulcer 6 intestinal adhesion 2 cholecystitis, terminal ileitis 2 chole-lithiasis 3 ileocaecal-tumor m. 2 gastritis 1 intestinal polyp 1 unknown 7 diverticulitis 1 chole-lithiasis 1 others 1 unknown 2 Total 33 * Total ' 20

105

Table III-4. Preoperative diagnosis of gastric anisakiasis.

Preoperative diagnosis Number Complications of (histological . cases diagnosis)

eosinophilic granuloma of stomach 2 peptic ulcer 37 ' 15 stomach cancer 31 3 gastric polyp 32 3 gastric tumor 32 chronic gastritis 6 2 duodenal ulcer 5 2 acute cholecystitis 2 1 chole-lithiasis 4 2 atrophic gastritis 2 pyloric stenosis 1 acute abdomen 1 106

Table 111-5. Preoperative diagnosis of intestinal anisakiasis.

Preoperative diagnosis Number of cases intestinal anisakiasis 2 acUte localized enteritis 12 eosinophilic granuloma of colon 1 acute abdomen 2 acute appendicitis 22 chronic appendicitis 1 ileus 9 abdominal tumor 4 intestinal canal adhesion 4 tuberculosis of ileum 1 right ovario-tubal inflammation 1 diverticulitis . 1 pyloric stenosis 1 duodenal polyp 1 chole-lithiasis 1 unknown until laparotomy performed 3

• ____, 107

Table 111-6. Period from disease occurrence (symptom complaint) to the operation.

Paroxysmal occurrence acute abdomen) 17 cases Within one month 6 One to three months 19 Three months to one year 13 More than a year 6 Unknown 31

92 cases

., 91 •C Z 5ii ge in 7 T '.e gi feee ma= 44 17 I-I .i.:3?sgfU3,:e3) .r.e118 3 :21 .-1 19 16 .,q CI® ',eV ez. 22 6 a' •'5 T eCD 15 6 t• .f- iq). 6 6 àl: .\' ' (ie) 6 oi 2 r 1 T 3 f 1 /5-Ifun 2 le 1 X s.., • el ii: ,,I W91-;KiLl)(19.1r231 II 1-1 ;ten 3 s 56 11(.21/ 2 4

Fig. 111-8. Main symptoms of anisakiasis (Ishikura, 1969). 1. Precordial pain. 2. Abdominal pain. 3. A feeling of plenitude in the precordial region. 4. A feeling of plenitude in the abdominal region. 5. Vomiturition. 6. Vomiting. • 7. Loss of apetite. 108

8. Emaciation. 9. General lassitude. 10. Heartburn, 11. Vomiting blood. 12. Hematochezia. 13. Anal blood flow. 14. Diarrhea. 15. Pleuralgia. 16. Notalgia. 17. :Jaundice. 18. Phyma in the precordial region. 19. Phyma in the abdominal region. 20. Symptom of . irritated peritonaeum. 21. No symptom. 22. Mass medical examination. 23. Discovered when opeating. 24. Autopsy performed after death. 25. (Note): Those on the left are gastric anisakiasis and those on the right are intestinal anisakiasis.

o €.%. s-N a 11 L Gil" 13 G14. g«,ek,a-à ! e m 0 8111 # • 1 c9 x 4 7■ ' C, (D * 33q 11 teneS fg. n 4 51. A W iri n 7c)

1 43 Z 1 111 25 E14 11.200 - + - * 11 - - 2 30 /I 1 'tT 11.000 •T + + * * im * - •- 3 18 ;Mt 1 T 10,800 - + + t 11 e -- 4 39 e 5 10 ,1,4*1 1 T Mk - - T + '11e e..* . 1/ V: i>5 - + 6 36Z#* Ti + - - .T if:ikft )1111, 1 -2 + 7 17 Z 1fie.tffi 1 ket 12.000 Z- * + - eT + p4 It 11 MTh1II- - + 8 19 Z11 1 T 7.400 ,11 - + - F + It hi 13.4 f1‘, + - 9 34 2 Ta 15,600 1L - + - 18T 4 1H1 t 114 - 10 24 Z 44 2 2 Tit 14.800 + ttte, + t-T + iM fl A + + 11 26 Z A II 1 Ta 13.800 t - 18F + a t 11 11-' A - 12 18 ZAlt 1 1111 12.800 + ti ",r4 + + 13 21 Z 4 I 5 TS! 11,400 + * - TA* - 8i8 N%1/Mieti - 14 22 1 *.CT 20,200 el ± - ti ,è t it m‘t + - 15 35 Z ;*!. 5 -FU 10.000 T h1t 1 7 = + + 16 19 A. 1 e 6. 400 * - -7 1k.A * ff .3 7 ,4 CE +

Table III-7. Clinical state of intestinal anisakiasis. 1. Case number. 2. Age. 3. Sex. 4. Occupation.

109

5. Number of days from pathogenesis to the first visit to doctor. 6. Subjective pain. 7. Leucocyte count. 8. Body temperature. 9. Vomiturition and vomiting. 10. Feeling of plenitude in the abdominal region. 11. Muscular defense. 12. Tender point. 13. Fur coating of the tongue. 14. Defecation stool. 15. Clinically diagnosed names. 16. Larval body found. 17. Macro. 18. Micro. 19. Induration.

Case No. .Occupation Subjective Body pain temperature 1 Factory manager Navel Normal 2 Agriculture Lower right Normal 3 Fishing industry Lower right Normal 4 Pit surface worker Lower right 5 Elementary school Lower abdomen Slightly high child 6 Housewife Lower abdomen Normal 7 Barber Navel Normal 8 Electrician Lower abdomen Normal 9 Factory worker Lower abdomen Slightly high 10 Agriculture & Lower abdomen Normal indus try 11 Fishing industry Lower abdomen Normal 12 Agriculture Stomach Normal 13 Electrician Lower abdomen Normal 14. Driver Lower right Slightly high 15 Fishing industry Lower abdomen Normal 16 Fishing industry Navel Slightly high

110

Case No. Tender point Defecation Clinically stool diagnosed names 1 Mackenzie's Normal Acute abdomen point 2 Mackenzie's Normal Acute appendicitis point 3 Mackenzie's Acute appendicitis point 4 Stomach Acute appendicitis . 5 Lower right Constipation Acute localized enteritis 6 Lower abdomen Normal Postoperative adhesion lieus 7 Lower right Constipation Acute localized enteritis 8 Lower right Normal Acute localized enteritis 9 Under navel Diarrhoea Acute localized enteritis 10 Lower right Normal Acute appendicitis 11 Under navel Normal Acute appendicitis 12 Mackenzie's Normal Acute localized point enteritis 13 Lower abdomen Normal Recurrent localized enteritis 14 Mackenzie's Normal Acute localized point enteritis 15 Lower abdomen Normal Intestinal anisakiasis • 16 Mackenzie's Normal Intestinal point anisakiasis 111

Table III-8. List of the affected parts in 92 cases.

Corpus ventriculi and Lesser curvature side 14' vestibulum ventriculi Greater curvature side 15\- 4 Unknown 15) 60 Stomach (65.2%) Pars pylorica 11 Unknown 5 Small intestine Distal part of ileum (more than 30cm 8 towards the mouth side from Bauhin's valve in the entire area of the ileum) 13 Caecum 5 28 Large intestine (30.4%) Colon ascendens 1 Rectum • 1 Others Pancreas Great omentum ]12-_ 4 Mesentery (3.4%) 112

Table 111-9. Affected parts.

Cases in stomach Fundus of the stomach' 1 Corpus ventriculi 17 Vèstibulum ventriculi 5 Great omentum 1 Unknown 9 Total 33

Cases in intestine Duodenum 1 Jejunum 2 Ileum 8 Caecum 4 Tissue around the ileocecal 3 region Colon 1 Unknown 1 Total 20 • •Ie 5:i " " 0 t 001 ru ,z 4 t e 18 7h {t. 11 et I: I 43 * * * * — 45cm 2 30 .1' * * * — * - * - m 3 17 -H- -H- * — — + + + so 439 + FF — * * * — PC) 5 10 * * + 20 6 3“- -H- — + ± — A 150 7 17 * + — * - - 8 19 Z -ef + + * + 10 9 34 Z * + * — — — * — 20 lo 24 Z **41- + * — — — 11 27 q- * + -H- + — * — — 6o 12 18 Z + ± — ± — — 30 13 21Z * ± — ± + —+ A 70 14 22 Z * *4- * * — + 30 I535 * -H- — ± — * — 70 16 19 * * — ± * — 4F — 70

Table III-10. State of intestinal anisakiasis in the abdominal cavity at the time of laparotomy. 1. Case number. 2. Age and sex. 3. Haemorrhages of intestinal serosa. 4. Opacification of intestinal serosa. 5. Edematization in the intestinal canal. 6. Necrosis of the intestinal serosa. 7. Intestinal canal cavity stenosis. • 8. Intumescence of lymphatic glands. 9. Necrosis of the lymphatic glands. 10. Abdominal dropsy. 11. Appendicosis. 12. Diseased region (mouth side from Bauhin's valve) 13. Distal part. 14. Jejunum. • 15. A Appendix is not removed.

114

Pathology

Àccording to the pathologic and histological conditions reported by Yoshimura (1966, 1967) and Ishikura et al. (1967), a high degree of edema and a marked infiltration of eosinophils in the submucosa can be observed commonly in both gastric and intestinal types. In the gastric type especially, • angiopathia, periangiitis, and the exudation reaction of neutrocytes, histocytes and plasma cells are observed, with the appearance of various foreign body giant cells around the larval body (in the process of necrotization). Generally, they appear as a form of abscess granuloma. In the intestinal type, pathological changes such as ulceration and necrotiza- . tion in the mucosa are not observed, and only rarely can a light degree of lymphocyte, eosinocyte and neutrocyte infiltra- . tion be observed. In the submucosa, edematization, separating of the fibrin and fibrinoid and haemorrhages are observed . as well as croàs section views of the larval body and helminthic route. In the tunica muscularis, fragmentation and . diastasis of the muscle bundle can be seen. In the serosa or subserosa, strong edematilation, separation of fibrin, heavy eosinophilic infiltration, activation of blood vessel walls and fibrose can be observed. Therefore, it is considered 'that in the intestinal type, an antizenic Teaction of a mainly phlegmonous type is presented. 115

As a result of a pathological analysis by Aizawa (1968) of the series of anisakiasis host reactions mentioned above, it was found that acute or chronic eosinophilic phlegmon of the stomach and intestinal submucosa and abscess or abscess granuloma are the lesions caused in the human body by anisakiasis. Moreover, Kojima et al. (1966) and Kojima (1966) classified the affected parts pathologically, histologically . and morphologically into (a) phlegmon type, (b) abscess type, (c) . abscess granuloma type, or (d) granuloma type, and analyzed the relation between these histological types and the conditions after symptoms appear. Referring to the results by Kojima et al., the histological picture of anisakiasis is a series of lesions which are interposed between the above- mentioned four histological types and include a migration type, and the above-mentioned four can be divided into two, one consisting of the former two types which are an exudative inflammation, the other consisting of the latter two types which are a productive inflammation. In the productive inflammation type, the diagnosis of histological identification becomes unavoidably more difficult when the larval body in the affected part becomes necrotized so that it is diagnosed just as a helminthic granuloma. However, a certain degree of analysis of the lesion is possible by special staining, and it is quite impressive to see the proliferation of histocytes or the 116

appearance of giant cells surrounding the remnants of the necrotic larval body or the cuticular sections. The various lesions mentioned aboveSenerally move from acute exudative inflammation to chronic productive inflammation as time passes, but it is difficult to consider them as a simple foreign- body inflammation due to the penetration of larval nematodes into the gastrointestinal wall. Some of the phlegmon type show the same or a similar picture of allergic angiobathia. Aizawa (1968) concluded that in the productive inflammation type there is definitely something connected with a specific host reaction which gib modifies the simple foreign-body inflammation, but the pathological analysis of the lesion is a problem for the future. Shiraki (1969) assigned 57 cases of human anisakiasis which he had examined himself to the four histological'picture types of anisakiasis from Kojima et al. (1966) mentioned above. As a result, there were 14 phlegmon type cases, - 25 abscess type cases, 12 abscess granuloma type cases and 6 cases of the granuloma type. Moreover, larval bodies or their remnants were detected in the lesions of 48 out of 57 cases. These 48 cases were divided into the following five groups according to the .state of the larval body:

Group I: Larval body fairly fresh - 7 cases. Group II: Slight degeneration observed - 5 cases. 11, Group III: Advanced degeneration or necrotization - 24 cases. 1 17

Group IV: Severely necrotized and the body cannot be recognized - 8 cases. Group V: Only moulted parts were observed - 4 cases. The relation between the larval body state and the histological picture ià as shown in Table III-11, and all of Group I in which the larval bodies were fairly fresh were phlegmon type, all of'Group.II for which they were slightly . 48 degenerated were abscess type in which a marked abscess was • formed around the larval body, Group III in which the degeneration and necrotizatiàn were advanced varied, with 14 abscess type, 6 abscess granuloma type and 4 granuloma type cases. In Group IV in which the destruction had advanced so far that recognition of the larval body shape wàs impossible, there were 4 abscess type cases, 4 abscess granuloma type cases and 1 case of the granuloma type, i.e. fewer abscess types and more abscess granuloma types than in Group III. The four Group V cases in which only Moulted parts were observed were 3 cases of the abscess type and 1 of granuloma type. The remaining 9 cases in which the larval body state could not be ascertained were 7 of the phlegmon type and 1 case each of àbscess and abscess granuloma types.

118

Fir 31 ? ___ ....._ Th m 11 _. DM (,î IV tf. i \V . ,-_:-_- fi il' P. -", ,n- tri: 1: K 11 xi/xi= "-10.. *e... 2.- ?fi P ..i M i t M it o) n• .c., . 7 7 1 4 ( 1 : 6 ) ( 5 : 2 ) ( 6: S) Il 5 la 3 3 ' 1

( 4 : ) ( 8 : 6) (3 : 0) (3 : 0) (1 : 0) s.ls,:): 7) 6 4 1 :0 1.-Igi ,15i4M I. ( 6: 0) (3 : 1 ) ; 1 : 0 ' t 9: I) u v 4 1 1 t3 t I : 3) (0: 1) (1 : C ) , 2: 4 ,

/ 5 24 8 4 9 ,,---,-(i (1 : 6) (a : 1 !15: 9) (6 : 2 ) ( 4 : 0 ( 7 : 2 ' ,3- :-:\-' ) ( )111 ( IV4(7)ae,G5C01 0.

Table III-11. State of larval body and the histological picture. 57 cases of anisakiasis or of a similar (?) disease. 1954 - 1968. 1. State of larval body. 2. Group I: fairly fresh. 3. Group II: slightly degenerated. 4. Group III: degenerated, destroyed. 5. Group IV: severely destroyed. 6. Group V: only moulted parts. 7. Helminthic foreign body not found. 8. Total. 9. Histological picture. 10. Phlegmon type. 11. Abscess type. 12. Abscess granuloma type. 13. Granuloma type. 14. Cases. 15. Numerals inside the brackets are the gastric cases and intestinal cases. 119

Aetiology.

By any account, the establishment of the lesion, i.e. the mainly aetiological and morphological occurrences of anisakiasis, is still a question here. Three items can be postulated for the cause of this: (I) The question of the predisposition of the organs. (II) Parasite and host factors and interrelationship. (III) Time of examination of lesion, i.e. an artificial cause. For these fundamental problems, we have to wait for future research. Investigations on the cause of anisakiasis occurrence are mainly conducted by using experimental animais such as dogs, rabbits, rats or mice. At'present, mechanical injury ›. due to the invasion and migration of these larVae, their metabolism products and in particular antigen-antibody reactions against the dead larval body constituents are considered to be the background for the establishment of the lesion. Although the reaction in these experimental animals is essentially different from the occurrence cause in the natural course of human cases, everybody can agree at least with the view that a sensitization reaction, i.e. an allergic reaction to an allergen which is largely the larval body or larval body constituents, plays an important role here. 120

Ishizaki (1967) considered the cause of helminthic diseases in general to be allergies and conducted a literature study. Moreover, Matsumura (1968) proved from their past research results that ascariasis is an allergic disease due to ascarid sensitization. Among studies on the cause of occurrence of parasitic diseases, particularly those on ascariasis, ascarid poisoning research has been conducted for many years, being commenced by Shimamura and Fujii in 1916, and later undertaken by Koizumi (1954) and his cooperating researchers. Although the occurrence of ascariasis was suggested to be an allergic reaction in the reports of these earlier workers, they still ascribed the reason to ascarid poisoning, so one is justified in thinking that helminthic diseases came to be discussed from an allergy standpoint.only recently. On the basis of the generally acknowledged opinion that anisakiasis is also an allergic reaction, further investigation from this standpoint is desirable in the future. In the above, we have briefly described the clinic, pathology and some of the reasons. for the occurrence of anisakiasis, and more time wilI be necessary for the analysis and conclusion of its aetiology or aetiological problems. 'Here we have only described the main reaction changes of the host body caused by this disease. 121

pimnmis

As mentioned earlier, it has not been very long since the disease called anisakiasis was first established. However, this does not mean that there were no cases of this disease earlier, rather we can consider that they were not recognized. under clinical diagnosis. A therapeutic method has not been established even to the present day. The reason is due to the difficulty of preoperative diagnosis as well as the pathognomonic symptoms of this disease. Since Anisakis larvae do not become adults in the human body, it is impossible to judge by the diagnostic method of fecal examination usual for ascarids or hookworms which infest the human intestinal canal. At present, there are no techniques for a certain diagnostic method other than to discover the larval body by resecting the lesional part of the affected area or to judge from a microscopic examination of the tissues in the lesional part. Ishikura (1968) considers the following items important for a diagnostic method of differentiating intestinal anisakiasis - from acute appendicitis or ileus: (I) consultation; (II) position and width of tender part; (III) X-ray 127

appearance; (IV) pyrexia, leucocytosis, interrelation of abdominal plenitude and muscular defence. Apart from these clinical diagnostic methods, studies of immunological diagnostic methods based on the reaction between antigen .extracted from larval bodies and antibodies has been carried out in the past few years. For instance, the method of diffusion in agar (Yoshimura, 1966; Suzuki, 1968), the precipitation reaction by superposition method (Taniguchi, 1965), the immuno-electrophoresis method (Morishita, 1966; Taniguchi, 1966; Suzuki, 1968; Yokokawa et al., 1968), the starch gel electrophoresis method (Suzuki, 1968), the erythrocyte agglutinatiori reaction which has the possibility of finding an important key to the diagnosis of anisakiasis patients (Kobayashi, Kumada, 1967; Kobayashi et al., 1967; Suzuki, 1969), the complement fixation test (Yoshimura, 1966; Kuipers, 1962; Merkelbach, 1964), intracutaneous reactions which are being applied practically to the diagnostic • method using the extract of Anisakis larval bodies or rearing liquid as an antigen (Taniguchi, 1965; Kobayashi, Kumada, 1967; Morishita, 19'65; Hàyasaka et al., 1968; Suzuki, 1969; Ishikura, 1969), separation and refining of the antigen material specific to Anisakis larvae as an immunological diagnosis of this disease (Suzuki, 1969), etc.. ' As can be seen from the above, there are quite a few immunological reports. 123

One of the problems which always occurs when an immune reaction is discussed is that of cross reactions, that is, the appearance of non-specific reactions based on the presence of a common antigen, and this has to be taken into consideration. However, Taniguchi (1965), Yoshimura (1966), Morishita (1966) and Suzuki (1968), etc. found common antigenicity in the cross precipitation reaction among each anti-serum which reacts to the antigens of man, dog and pig ascarids or of Anisakis larvae. This prevents the establishment of an immunological diagnostic method for anisakiasis. Taniguchi (1965) investigated the reaction patterns of the antigens of Anisakis larvae,- dog and pig ascarids against the anti-serum of Anisakis larvae which were parasitic in the abdominal cavity of Trachurus japonicus and reported that although there was a common antigen in the above three, it was characteristic that the antigen of Anisakis larvae possessed a reaction pattern intermediate so to speak between the other two.

Yoshimura (1966) also cOnducted an antigen analysis by the cross reactions between the serum of various animals and four kinds of antigen by the agar gel diffusion method (Table III-12). That-is, Anisakis larvae were orally administered into guinea pigs once or repeatedly, and other dog and pig ascarid mature ova were administered into guinea

124 •

.•-s cr: .k!s3q1 F„,.;:JP, ® l5) .:qt x.—li ,%: ,1:

M1 * + _ ± M2 + ± + + M3 + + _ — M4 + — — ± M5 — — _ — (b , • Mc — — — — 07 ±, --/- .7„ .. RI ± _ ± (..:1e.a. R c — — — —

MI + + e — M 2 + + + ±

(esiE5 - 5- MI + + + + M 2 ± ± — + ;,:.D.5)7. D — ± + ± • D:). c : Table 111-12. Cross reaction results between the serum of various animals and four kinds of antigen by the agar gel diffusion method (Ouchterlony) 1. Antigen. 2. Antigen of Anisakis larvae. 3. Antigen of dog ascarid adults. 4. Antigen of dog ascarid larvae. 5. Antigen of pig ascarid adults. 6. Serum. 7. Anisakis infection. 8. Control. 9. Anisakis infection. 10. Dog ascarid larva migrans. 11. Pig ascarid ova administration. 12. Dog which has dog ascarid adults. 13. M: guinea pig, R: rabbit, D: dog, c: control. 125

pigs in the same way and blood was removed three to eight weeks later, and then the appearance of the precipitation lines for the Anisakis larvae antigen, dog ascarid adult and larvae antigens, and pig ascarid antigen against each Serum from_these animals were observed and the results were summarized as in the above Table. In the Anisakis larva .migrans, a fairly clear reaction against the same kind of antigen was observed and the precipitation line even appears . againSt the antigen of some dog or pig ascarid adults and also for other larval nematode migrans the appearance of a cross reaction cannot be excluded. A common antigenicity with ascarids can be presumed in Anisakis-lie larvae as in the other kinds of nematoda. Morishita (1966) •reported similar results, and Suzuki (1968) observed the appearance of cross reactions among Anisakis larvae, human or.pig ascarids in all cases of intracutaneous reaction, agglutination- reaction of erythro- cytes, agar diffusion method, etc.. On the other hand, Yokokawa et al. (1967, 1968) investigated whether any specific antigen constituents exist in the extract from Anisakis larvae by the dmmuno-electrophoresis method, and they observed one characteristic precipitation line left in the absorbed Anisakis larvae anti-serum sensitized by the antigens of pig and dog ascarid adults. However, one will appreciate that if the specific

.1.26

nature of the antigen is the main question, this is not answered sufficiently yet as far as the establishment of a specific immunological method of diagnosing anisakiasis is concerned. Recently, an intracutaneous reaction (cutireaction) in which extracts from Anisakis larvae or their excrement, secreted material or Moulting fluid are the antigens, and an erythrocyte agglutination reaction are being tested as measures in a search for the facts of Anisakis disease infection. Taniguchi (1965, 1966) and Morishita (1966) conducted a cutireaction for one anisakiasis patient, ten Gifu University class members, and 58 students of the Medical Science Department in Gifu Prefecture, and found 1, 2 and 20 people positive in each case respectively. Moreover, Taniguchi (1966) and Morishita (1966) conducted a cutireaction e_ for a 23 year-old man who was diagnosed as having peptic ulcer in February 1966 and a larval body was found in the polyp of the part excised from him at the time of laparotomy, by using the antigen of Anisakis larval body which was obtained frm Trachurus japonicus, and after 15 minutes they obtained the positive results of a 35 x 50mm flare; and 17 x 25mm wale. When Kobayashi and Kumada (1967) conducted cutireactions for 239 average adult people in a rural village

in Shizuoka Prefecture by using two sorts of antigen, that is,a larval body extract and the rearing liquid, the positive percentage was 9.2% (22/239) for the larvai body extract antigen and 20.9% (50/239) for the rearing liquid antigen, and the sensitivity was higher in the latter. In an investigation of the'density of tannic acid'and antigen in the erythrocyte, agglutination reaction which was conducted at the same time, the anti-body production in sensitized animals was faster during oral infection than during infectio n in the abdominal cavity, and the percentage of the buried cutireactions and erythrocyte agglutination reactions which agreed qualitatively was approximately 70%. When Ishikura (1969) examined the relation between the outbreaks of intestinal - anisakiasis and the intracutaneous reaction of healthy people, the positive percentage increased in the sequence Gifu, Shizuoka, Niigata, Hokkaido (Fig. III-9). Also when Suzuki (1969) inoculated 0.54cg of refined antigen to five anisakiasis patients who had had an operation within a year, they were all positive. Moreover, when 100 adult people from Iwanai Town in Hokkaido where anisakiasis is prevalent and 40 long time in-patients of a mental hospital in Aizu Wakamatsu City were examined, the positive percent- 52 age of the former was 60% and that of the latter was 0%. At the same time, when an indirect erythrocyte agglutination reaction was conducted for the serum of these five cases of anisakiasis patients, all were positive except the one case in which the larval body was . largely destroyed. i28

Moreover, Kobayashi et al. (1968) and Kobayashi (1969) examined two types of antigen for use in intracutaneous reactions, one extracted from larvae (SON antigen), and ES antigen, the main constituents of which are secreted materials ar excrements in the larval rearing liquid. The method was to inject 0.2 mi of each antigen (protein density 30p..g / m, ) intracutaneously, and the test was judged to be positive if a wale ( 9 mm) or flare ( 20 mm) appeared after 15 to 20 minutes. In the investigation results for Shizuoka and Ehime prefectures, out of 769 people the positive percentage was 4.7% for SON antigen and 10.5% for ES antigen. The positive percentage increased with age, being high in people over 30, and it was higher in men . than in women. These results were common to both antigens (Fig.

III - 10). These results of Kobayashi have the same tendencies as the results of Yoshimura (1966), Otsuru and Koyanagi (1966) and others in which percentages for different ages and sex were obtained from'Patient cases. Furthermore, for both

. antigens, the positive percentage of intracutaneous reactions of people who have the habit of eating various marine fish or cuttlefish raw was much higher than for people without such a habit, and the reaCtion appeared more often in people with• an allergic diathesis. When Yoshimura (1966) studied the intracutaneous reactions of rabbits which were sensitized with antigens of • 129

[I II 9 Aik

Fig. III-9. Percentage of positive intracutaneous reactions in healthy people. 1. Gifu Prefecture. 2. Shizuoka Prefecture. 3. Niigata Prefecture 4. Hokkaido Prefecture.

• Ï-G c=3 e- s

- à Er/ ® (i)

E s

• 3,,

• • • • • 2C •

Fig. III-10. Comparison of positive percentage for two sorts of antigen in different age groups and sex. 1. Positive percentage. . 2. SON antigen. 3. Men. • 4. Women. 5. Average. • 6. Age. 7. Es antigen 130

Anisakis-like :larvae and rabbit or dog ascarid adults by Evan's blue method, there were many things to,be investigated with regard to cross reactions. Suzuki (1969) attempted the separation and refining of the specific antigen substance for the Purpose of solving the problem of the existence of -a common antigen substance which is lacking in the above-mentioned immunological diagnostic method for anisakiasis and for the purpose of establishing a certain and specific diagnosis. That is, using the Starch gel electrophoresis method, he migrated the substance extracted from Anisakis larvae and that from ascarid adults at the same time and performed a fractionation of the coarse antigens, and found that the water soluble protein constituents of which the two were composed were quite similar. By further fractionation of the fractionated extracted constituents by the agar gel diffusion method, he found that the 6th fractionation of Anisakis extract reacts specifically to the Anisakis anti-serum. By collecting the constituents extracted from this fractionation and ' migrating them again by starch gel, considerably refined constituents were obtained. He collected the target fraction by DEAE-Cellulose, and refined the antigen by using a pre- parative disk electrophoresis apparatus. As fundamental .biochemical studies oranisakiasis rather than its immunological diagnosis, Okuno (1968) 131 • investigated amino acid composition by thin layer chromatography, and Kawai (1968) investigated free hieer fatty acids by gas chromatography. As a result, there were 11 common kinds of amino acid in all pig ascarid tissues, and alanin, phlorin, glycine and glutamic acid . were detected as heavy spots. 14 kinds of amino acid were found from Anisakis larval bodies and the're were heavy spots àf alanin, glycine and phlorin. There were 14 kinds of free higher fatty

acid from 010 to 018 detected from pig ascarid tissues. They were in quantity mostly 0 16 and 0 18 in both AnisalKis larval body and pig ascarid tissues, the unsaturated higher fatty acids being more plentiful. In order to apply such results in practice, more precise research will be necessary. An outline of the 'clinical diagnosis and immunological diagnosis methods of anisakiasis published by February 1969 was mentioned above. More time will be necessary before any of these diagnostic methods may be applied practically with diagnostic value, and the earliest practical utilization of at least the immunological diagnostic method is desirable, since the method is an important key to understanding the sensitization of this disease or the actual details of the infection. 132

Therapy

As with the diagnostic methods, there has been no certain method of therapy either. In the serious gastric type cases, the quickest way is to excise the lesional part, but the minor cases will recover with about two weeks of rest. As for the intestinal anisakiasis, moreover, we should • concentrate on conservative medical treatment in order to guard against exudative inflammation, considering the cause of occurrence to be an allergic reaction, since the disease breaks out without larval invasion into tissues. Anti- allergic medicines, antiphologistric non-steroid medicines, or hormones of the steroid group, etc. are considered to •be the main remedies for such treatment, but there are no definite reports concerning the applicational value of these 54 remedies. In a.ny case, since a diagnostic method has not been established, we must consider thatit is difficiat to devise a therapeutic method at this stage. Therefore, the establishment of early preoperative diagnosis will be the primary problem.

Summary

In the above, we first summarized the historical 133

process up to the time when the notion of anisakiasis was established, and then the epidemiology, clinic and pathology, and diagnosis and therapy. As a result we had to acknowledge that there are many things yet to be solved in each item. In particular, further investigation will be necessary in analyzing anisakiasis from the pathogenetic or aetiological standpoints. There are too many factors in anisakiasis to end with the phenomenological conclusion that this disease is a simple infection.disease caused by the migration of Anisakis larvae (mainly type 1) into the human body. If the investigation is conducted just with regard to the reason for establishment of the infection, there are many internal and external factors affecting the pathogenicity of this disease, such as the conditions on the larval body side (differences in action motivation of each larva, number of infecting larvae, larval body constituents or secreted substances and excrements) and the conditions on - the host body side (constitution of patients, and other factors). As mentioned earlier, the actual parasitism and detection of this larvae does not necessarily bring the occurrence of anisakiasis, and this substantiates the theory that the conditions of the infecting larvae or the host body are closely connected with the outbreaks of this :disease. It might be possible to find some correlation by investigating this disease from the. standpoint of host-parasite 134

relationships: Moreover, the variation of the infective behaviour according to the individual or area is a question

yet to be solved. We will conclude here by saying that ' we have understood only the fact that some complicated mechanism is involved in the establishment of infection of this disease as in the case of some other'bacterial or helminthic diseases. 135

• IV. EXPERITUNTAL ANISAKIASIS 55

Si nif c ano e of Experirnental Anisakiasis

The purpose of anisakiasis infection experiments is to investigate the fact,ors of both host side and parasite side which are involved in the establishment of anisakiasis. That is, with infection experiments on experimental animais as dogs, rabbits, cats, rats or guinea pigs, the host such side similarity to human cases is investigated through their behaviour to infection and their pathologic histological pictures, the excrements and secreted material of larvae and foreign proteins of the dead larval body are presumed to be parasite-side factors, and from an oVerall view of these, attempts are made to solve the cause of anisakiasis infection occurrence.

One of the parasitological advances after the war is the establishment of the connection with the helminthic allergy area. During the last twenty years, immuno-serology or various other techniques have been developed, and the establishment of immuno-chemistry has had an important influence on the parasitological field. And in the clinical field also the symptoms are considered to be due to an 136

allergy and attempts are being made to prove this by animal experiments and clinical research. Many parasitology researchers have already demonstrated th u allergic analysis or proof in ascarids, hookworms, Wuchereria, Gnathostoma and Bilharzia, and the cutireaction (intracutaneous reaction) employing an allergic reaction is already established as a practical diagnostic method. The pathological and histological investigations in human cases to date or the results of animal experiments on infection by Anisakis larvae strongly support the participation of a localized allergy in the cause of anisakiasis occurrence. That is, in the earlier stage, it is considered that the sudden- and acute lesion. (allergic inflammation) in a local area is caused by the mechanical injury due to the larval migration, metabolism products (secreted material, excrements) when the larvae are alive, and by the necrotized remnants of the dead larvae. In this chapter, we are going to describe mainly the host reaction behaviour when experimental animals are infected by these larvae as well as the relationship with factors of the parasite side. Incidentally, there are many reports of experimental anisakiasis in Japan. For instance, reports in which the reason for occurrence of anisakiasis is considered to be a local allergy (Usuya, 1964; Usuya and Shinno, 1965; Usuya, 11 5 '7

1966; Otsuru at al. 1966; Otsuru et al., 1966, 1967; Koyana4, 1967; Koyanagi, 1967; Shinn°, 1966; Kikuchi et al., 1967; moue et al., 1968; Ishikura et al., 1968; Ishikura, 1968; Ishikura, 1969) - these are the greater portion of all the reports; reports in which the conditions of larval migration into the host body (mainly the larval distribution in the host body or the detection of larvae) are discussed (Hirao and Yamaguchi, 1964; Yokokawa et al., 1965; Isobe, 1965;

Yoshimura, 1966; Okumura, 1967; Matsuoka and Usuya, 1966; Nagase, 1968; Shiraki, 1969); reports which attempt to investigate the cause of occurrence of anisakiasis from the factors of both host and parasite sides (Asami et al., 1964,

1965; Asami et al., 1965; Asami, 1966; Inosnita and Asami, 1967); and reports which attempt to clinically investigate the state of blood and serum at the time of anisakiaSis infection by using experimental animals (Matsuoka, 1965, 1966). Although these reports are divided roughly as above for convenience' sake, strictly they cannot be divided as they are in fact related to each other. Furthermore, the range of experimental hosts is wide, the experimental animals used ranging from dogs and rabbits to domestic fowls, bullfrog (Rafla esculenta) and silver carp. Abroad, Myers (1963) and Kuipers (1964) attempted to analyze the pathogenesis of this diseàse by using guinea pigs and rabbits respectively. 138

Here, by summarizing and considering the experimental results in the above, we would like to point out the significance of experimental anisakiasis and discuss the directions o f food hygienic research concerning anisakiasis in the future.

Mainl Concernin the Localized Allergic Reaction

Usuya (1964) infected rabbits with single or repeated oral administration of Anisakis-like larvae, and investigated the histological reaction around.the larval bodies by performing autopsies at different time intervals. In rabbits the histological reaction around the larval bodies was conspicuous, and the reaction was macroscopically and histologically more marked in the repeated infections. Larval migration through the alimentary canal wall was only observed in rabbits which were repeatedly infected. Moreover, in the repeatedly infected rabbits, the formation of an eosinophilic granuloma was observed around the necrotic remnants of a dead larva and the participation of allergic factors was assumed. Usuya and Shinno (1965) also conducted experiments . on oral administration of Anisakis-like larvae collected from Trachurus jap.onicus and Coryphaena hippurus in 50 dogs, 139

4 cats and 24.rabbits, in order to clarify the cause of pathogenesis of anisakiasds infection. The experiments were carried out using three methods, i.e. single oral administration of larvae, repeated oral administration and burying larvae in the stomach wall after laparotomy, and the following results were obtained by subsequently performing autopsies and macroscopic and histological examinations. ' 1. In dogs to which a single oral dose was given, the larvae had already penetrated after one hour, and the presence of larval bodies was verified even after 50 days. Up to 15 days afterwards larvae were observed to be still definitely alive. After this, larval bodies were not found in the alimentary canal but the formation of small granulomas was observed in the great omentum. 2. The histological changes in dogs which had single infections were generally slight. 3. Some dogs vomitted 10 to 30 minutes after oral administration and did not become infected. 4. The reaction in the region of larval invasion became stronger in the order rabbits, dogs, cats, and in rabbits marked eosinophilic infiltration was often observed around the larval body.

5. When repeated infection was employed, the local reaction was much stronger than in the case of single 57 infection. 140

Furthemore, Usuya (1966) infected 7 dogs usint; repeated oral administration of larvae. In macroscopic autopsies of the alimentary canal, saine larvae were observed to have already penetrated the stomach wall one hour after the administration and haemorrhagic spots were observed in the area penetrated, and many of the larvae were boring into the surface of the stomach mucosa, and especially into the small intestine mucosa, with the anterior part of their body. In the experiment using 4 cats, these haemorrhagic spots were not observed, but the larvae were alSo penetrating as in dogs. In rabbits the haemorrhagic spots were strong over a wide area and the larvae were dead. The histological changes were strongest in rabbits and marked eosinophilic infiltration was observed around the larvaL bodies after 10 days. In dogs the infiltration of neutrocytes was only observed in the earlier stage, and in cats slight infiltration of neutrocytes Was only observed'in the areas penetrated by the larvae. In dogs and rabbits which were given repeated doses of larvae, the changes were generally strong compared with those which received a single dose, and more eosinophilic granulomas were formed. From the above results it was found that dogs and rabbits are easily infected by the Anisakis-like larvae which infest marine fish, and typical eosinophilic granulomas can sometimes be formed in the stomach wall even by a single 141

infection, and even in the same alimentary canal the reaction phenomena vary from the stomach to the intestine. Moreover, Usuya (1966) conducted research centered on the pathologic, histological problem by using 39 young dogs, 14 rabbits and 4 cats. First of ail he carried out single or repeated ,oral administration of Anisakis larvae collected from the abdominal cavities of Coryphaena hippurus, Trachurus japonicus and Pneumatophorus japonicus to the above experimental animals. After being infected, autopsies were performed on the animals at different time intervals, and he Observed the migration of the larvae into the animal body, their viability and macroscopic changes and the histological changes of the areas penetrated by the larvae. Tables IV-1 to IV-4 show results of the sites of larval migration into the body and time of survival of the larvae. According to these, in dogs the larvae migrated to the stomach cavity and stomach wall one hour after infection, and to the stomach wall, intestinal wall, great omentum and even to the mesentry after three hours. The administered larvae were almost all dead by 15 days. In the case of cats, the larvae were observed in the surface of the gastrointestinal wall mucosa for up to three days afterwards. In rabbits, larvae assembling in the stomach wall and ' abdominal cavity,were detected, but they were all dead ten days after infection.

14 2

..._ ® @) ® E Ji. in ,? ".e .-"-" ( m ) llt '., • 2. it» -C.7-- 0 .1'

3 30 • 1 14 30 • 19 32 30 1 • . 4 2 33 30 • 6 • 5 1 3 12 30 • 8 11 2 5 li 20 1 • 8 16 20 1 • 12 1/ 20 2 • 1 • GI • :fl u l

Table TV-1. Recovery of larval bodies from dogs infected by Anisakis-like larvae. 1. Time elapsed after infection (hours). 2. Number.of dog used. 3. Number of larvae administered. 4. Number of larvae recovered. 5. Stomach. 6. Small intestine. 7. Wall. 8. Cavity. 9. Abdominal cavity. . 10. *Haemorrhagic spots appeared.

143

c) ;0- e E, iiRem ,k c3it, ,,D ‘egiiç 12- UieVe) ey {4e •O g ( 8 ) Mk M2 CM (spe Om (ee 4 1 Ii 30 3 • 1 4 20 11 • . 1 ( D ) {192 30 2 • 2 15 ' 3 3 30 11 • 4 • 5 20 4 • 2 1 2 - 20 1 5 0 1 1 t22 20 1 6 8 30 2 1 2 6 20 1 7 { 25 20 1( D ) 20 2 8 • 23 20 5 9 9 20 1

10 30. 1217 20 10 20 1 - 15 124 20 1( D ) 2 ( 1 D) 6 ( D ) J 18 30 5 ( 4 D) 20 126 30 • 25 27 30 30 28 30 3 ( D ) • 40 31 30 ' 2 ( D ) 50 30 30 1 ( D ) ŒD • ttatuei ut D : yte

Table 1V-2. Recovery of larval bodies from dogs infected by Anisakis-like larvae 1. Time elapsed after infection (days) 2. Number of dog used.' 3. Number of larvae administered. 4. Number of larvae recovered. 5. Stomach. 6. Small intestine. 7. Wall. 8. Cavity. • 9. Abdominal cavity. 10. *Haemorrhagic spots appeared, 11, D: Died.

144

0 (g) EJ 4g' -)• '_É. e fee'" Iffl% e-- .r. 14; e ei + @ in ( 6 ) am (24.2 (70 1 1 20 2 2 2 20 1 3 3 20 4 30

Table IV-3. Recovery of larveal bodies from cats infected by Anisakis-like larvae 1. Time elapsed after infection (days). 2. Cat number. 3. Number of larvae administered. 4. Number of larvae recovered. 5. Stomach. 6. Small'intestine.. 7. Wall. 8. Cavity. 9. Abdominal cavity.

0 G !e: 4D:a cr) le. f rt '4, IS'," ...■ .t! tt.,1M It ■I ei. /,/ i ■ .., CO Vi • ; , (e) r

C r?- 2 2 30 3• -10 6 30 5( 0). 1 (01 15 5 30 3 ( D ) 1 (0f 20 4 30 30 3 30 50 1 30 2 ( 0 ) 1 ■ o:

• D : eE

Table 1V-4. Recovery of larval bodies from rabbits infected by Anisakis-like larvae. 1. Time elapsed after infection (days). 2. Rabbit number. 3. Number of larvae administered. 4. Number of larvae recovered. 5: Stomach. 10. *Haemorrhagic 6. Small intestine. spots appeared. 7. Wall. D: Died. 8; Cavity. 9. Abdôminal cavity. 14 5

On tha other hand, Otsuru (1965) tried to analyze the cause of pathogenesis of granulomas in the digestive system from the allergic standpoint, by inserting 30 Anisakis- like larvae collected from Scomber japonicus into the abdominal cavities of dogs and rabbits with subsequent autopsies and histological examination. As a result of the autopsies, the ratio of larval penetration according to • regions in dogs was: Stomach : Intestine = 1 : 1; and in rabbits it was 17.8 : 1. With regard to invasion into the stomach or intestinal wall in rabbits, it was found that in the sensitized group it was about 1 / 2 of that in the comparison group, the invading percentage decreasing when they are sensitized.. Moreover, as a result of histological examination, in the comparison group of rabbits, phlegmon accompanied by pseudo-eosinocytes and strong haemorrhaging occurred mainly 24 hours after the oral administration, and 72 hours after, a slight degree of eosinocyte infiltration and small abscesses appeared in all cases, and after a week foreign body giant cells appeared in one case only, whereas in the sensitized group, the infiltration of cells, mainly eosinocytes, occurred after 24.hours, after 72 hours the formation of giant abscesses were observed, and degeneration of the larval.bodies occurred at a relatively early stage, and it was found that in the sensitized group the cell 146

reaction commences rapidly in earlier .stages compared with the non-sensitized group and an appearance almost identical to that in human cases was presented. Moreover, Otsuru et al. (1966, 1967) acknowledged the possibility of allergy participation as a host-side condition of anisakiasis, and in order to reinvestigate the parasite-side factors, they performed experiments concerning the influence of the metabolic products of the larvae in the earlier stages and of the foreign protein of degenerated • necrotic larval bodiès in the later stage. The sensitization method was carried out by administering 30'live larvae into the abdominal cavity of rabbits two weeks before the various experiments. For metabolism products, larvae were reared with a ratio of one larva per 0.1 cc sterile isotonic sodium chloride solution with antibiotics, and 0.1 cc of the rearing fluid taken either before, during or after the molting was injected intracutaneously in the dorsal regions of both sensitized and non-sensitized groups of rabbits, and two days later autopsies were performed. As a result, eosinophilic infiltration was observed in every case. Eosinophilic abscesses were formed, and haemorrhages and edemas appeared particularly for the combination of sensitized rabbits and rearing fluid taken during molting. Moreover, when 0.1 cc of crushed larval bodies was injected into the stomach wall submucosa of sensitized and 147

non-Sensitized rabbits and autopsies performed after three and seven days, eoSinophilic abscess formation was observed in each case. In the sensitized group, the eosinophilic infiltration and abscesses were pronounced and (the lesion was large,-whereas in the non-sensitized group, the infiltration of pseudo-eosinocytes, histocytes and foreign body giant cells Was strong and the lesion was small and foreign- body like. From the above results, it was concluded that on the parasite side the secreted material, excrement ( in earlier stages) and necrotic remnants of dead larval bodies (later stages), and on the host side the condition of being sensitized by the repèated consumption of these larvae, play important roles as factors in the pathogenesis of anisakiasis. Koyanagi (1967) infected 65 rabbits and 14 yoUng dogs with Anisakis larvae collected from Scomber japonicus. The purpose of his experiment was to look for an allergic nature in the cause of pathogenesis of anisakiasis, and the infection was carried out by the three methods, i.e. oral administration of larvae, intracutaneous injection of the larval rearing fluid in rabbits and injection of crushed larval bodies into the stomach wall. The oral administration was carried out after dividing the experimental animals into sensitized and non-sensitized groups. By performing 148

autopsies at different time intervals, they were examined macroscopically and histologically. Some of the results are shown in Table IV-5. It was fouhd that in the non- sensitized group with oral administration there was a marked difference_between rabbits and dogs, especially with regard to the amount of stomach wall invasion, being twice as much in rabbits as in young dogs, and with regard to the part invaded, in rabbits it was mostly the stomach, whereas in young dogs, it was almost equally divided between stomach and intestine. The histological reaction was slight in both animals. When rabbits sensitized by inserting 30 live larvae into the abdominal cavity by performing a laparotomy were given an oral administration, a high degree of eosinophilic infiltration,.edeffias and phlebitis were observed even in the earlier stages and after one week large eosinophilic abscesses were formed, of a similar state to those in human cases. On the contrary, in the non-sensitized group only slight eosinophilic infiltration was observed. The amount of larval invasion in sensitized rabbits was about two-thirds of that in non-sensitized rabbits. Moreover, Koyanagi (1967) divided the rearing fluid of larvae into three types taken before, during and after the molting, and injected them into sehsitized and non- ' sensitized rabbits for the purpose of ascertaining whether the secreted material or excrement of larvae or the larval

:f v't r.n> ' 7? 1è 7 4 -s-' S•X 41/ 4.":4 p., .4 44 1 W 'I-

' ./ n t:,' _. * • ,.,. 1 _ _ + + _ _ _ _ fa el i_ '.?4 i rj: 4 6 + + -k + - - - ± t M Or 1- 81 .+ - t + -+ i -± fT 8 1.

- - - *04.q Y 4,9,57f re, 47 _t. f -11- W + + - ± 14 e.8 + o W+ 48 ± - if + - ± *TEA + 22 + ± + + - - - + ' 32 + + ± ± - - - + fY 2/1 7 72,3 m 82 + - + + + + - + ff e.4.§)

61 ± - - ± ± - + - 3-,_ li (Is) :F 83 ± - - ± ± - - - J4 EA C> + 84 + -- -- :■-• ± — — --

91 f - - - + ± .i...1. + 2)$ 92 k - - + -‘ i- yi: +-I + - 1- ± t'i. n

- I: - .... 4-: 1 11 971 ± - - ± - - k - vl 0 ± 103 + - - - - - SE SE .;...1. 4-

Table 1V-5. Experiment of oral administration of 30 Anisakis larvae to rabbits. 1. Histological state of non-sensitized group. 2. Time until the performance of an autopsy. 3. Rabbit number. 4. Eosinocytes. 5. Pseudo-eosinocytes. 6. Haemorrhages. 7. Edemas. 8. Phlebitis. 9. Fibrinoid degeneration. 10. Giant cells. 11. Abscesses. 12. Larval body. 13. Degree of lesion. 14. Clear. 15. Degenerated. 16. Partially degenerated. 17. 24 hours. 18. 48 hours. 19. 72 hours. 20.- 1 week. 21. 2 weeks. 22. 1 month. 150

molting could •be the sensitizing cause, and he obtained the following results (Table IV-6). That is, there were no marked changes according to macroscopic observation. However, histologically a slight to high degree of eosinophilic infiltration had appeared in each case, and the degree was in the order sensitized, during molting> sensitized, after molting> nonLsensitized, during molting > non- sensitized, after molting> sensitized, before molting 7,-> and it was deduced that non-sensitized, before molting, there is some relation between molting and infection. In the results of the injection of crushed larval body into the stomach wall (Table IV-7), it was observed by autopsies three or seven days after the injection that abscesses were formed in both the sensitized and non- sensitized groups, but in the non-Sensitized group many pseudo-eosinocytes or foreign body giant cells were observed, whereas in the sensitized group there are many eosinocytes and the edematization was strong, which is similar to human cases. From the summarized results of Koyanagi, it will be concluded that the pathogenesis of anisakiasis is mainly caused by an allergic nature due to the frequent consumption of Anisakis larvae.

Shinno (1966) buried Anisakis-like larvae which had been collected from the abdominal cavities of Trachurus

151

japonicus and 2neumatophorus japonicus and soaked in hot water for a moment, in the alimentary canal wall of young dogs and rabbits. The details are shown in Tables IV-8 to IV-11.

.i- f '-.; 1 ;' ;ti. k % 1if IES M. P?f, c9 . ® 4 AZ IZI CI q

C), 5 * + + + - + 0 n + + + - + 66* 67+ + - - - + 0 15+. + + - - + n 75 + + + + - . + M + + + - - -1- (:) 60 * * + + + -* ( t: A )- C) c 68 * * + + + * 69 * + * + f f1-1:7i;4) 0 m f + + - - * 79U + ± - - * 102 -e- + - - - + (a, + -H- ± + 70* 71 * + + ± ± * 0 89+ + - - + + 'mkcza (ù + + - - it 77* W * + * ± ± * 103± ± - - - ±

Table IV-6. Individual rearing of Anisakis larvae. 1. Rearing fluid. 2. Sensitization. 3: Rabbit number. 4. Eosinocytes. 5. Pseudo-eosinocytes. 6. Haemorrhages. • 7. Edemas. 8. Phlebitis. 9. Degree of lesion. 10. Performed. • 11. Not performed. 12. Before molting. 13. During molting. 14. After molting. 15. Abscesses. 15 2

a) 'err e4 ir 0.)

Table IV-7. Experiment of injection of crushed Anisakis larvae into the stomach wall of rabbits. 1. Histological state in sensitized and non-sensitized groups. 2. Time until the performance of an autopsy 3. Rabbit number. 4. Eosinocytes. 5. Pseudo-eosinocytes. 6. Haemorrhages. 7. Edemas. 8. Giant cells • 9. Phlebitis. 10. Size of lesion. 11. days. 12. Sensitization. 13. .PerforMed. 14. Not performed.

153

Table IV-8. Experiment of granuloma formation in the 62 stomach wall of dogs.

No. Region Number Method ' of days before ex aminat ion

1 Subserosa 7 1. Larva buried catgut suture) 2. Larva buried unsutured places 3. Catgut suture only 4 4. Larva buried catgut suture ) 9 Submucosa 7 1. 2. Larva buried catgut suture 3. 5 places 4. Larva buried silk suture 5. Silk suture only

12 Submucosa 1 5 Larva buried catgut suturel 3 places 3. Catgut suture only I 14 Subserosa 20 1. Larvae buried catgut suture (3 larvae) 2 places 2. Catgut suture only J. Subserosa 7 1. Extract from larvae injected intravenously three times. 2. Three larvae buried 21 days after the first injection. 015 Subserosa 1 7 1. Larva buried twice with six-day interval. 2. Three larvae buried in one place catgut suture 016 Subserosa 22 1. Larva buried twice with six-day interval. 2. Three larvae buried in • one place catgut suture

014 Subserosa 10 1. 30 larvae administered orally. 2. Three larvae buried after a week catgut suture

Note: 1. Larvae collected from Trachurus japonicus and Pneumatophorus japonicus were used. 2. Larvae soaked in hot water for a moment were buried. 154

Table IV-9, Experiment of granuloma fOrmation in the 6 3 intestinal wall of dogs.

Region Number Method of days before examination

Ileocaecum 7 1. Distal part of (subserosa) ileum. - 2. Caecum. Larva 3. Colon buried ... catgut suture ascendens. Ileocaecum 7 1. Distal part of (submucosa) ileum. 2. Caecum. Larva 3. Colon buried ... catgut suture ascendens. Ileocaecum 1 5 1. Distal part of (submucosa) ileum Larva buried ... catgut suture 2. Caecum catgut suture . only 3. Colon ascendens Larva • buried ... catgut suture Ileocaecum 7 1. Ileum, caecum, and stomach colon ascendens. wall 2. Three larvae buried in one stomach wall location. 3. Ten larvae in great omentum. Colon 1. Extract of larvae injected intravenously ascendens • three times. (subserosa) 2. Buried 21 days after the first injection. 1 55

Table IV-10. Experiment of granuloma formation in the stomach 'wall of rabbits.

No. Region Number Method of days before xaminati

101 Subserosa 7 One larva buried in each 103 Subserosa 7 / of three places catgut suture 105 Subserosa 7 106 Subserosa 10 107 Subserosa 15 Three larvae buried in 108 Subserosa 20 one place catgut suture 109 Subserosa 30 . . . 110 Subserosa 50 }

04 Subserosa 20 1. 20 mg steroid, injected intramuscularly daily for seven days. 2. Three larvae buried in one place the next day.... catgut suture

.06 Subserosa 18 1. Buried twice with 7-day interval. 0 7 Subserosa 27 1 2. Three larvae buried in one place catgut suture 0 14 Subserosa 10 1. Extract of larvae intravenously injected three times. 015 Subserosa 20 1 2. Larvae buried 21 days after . the first injection. 111 Control 50 ' Catgut suture only.

112 Control 50 Silk suture only. 15 6

Table IV-11'. Experiment in which larvae were depositied 64 in the abdominal cavity.

No. Region Number Method of days before examination

(Dog) 3 Basal part of 7 50 live larvae. mesentry.

4 Basal part of 1 5 50 live larvae. mesentry 5 'Basal part of 7 50 dead larvae mesentry and 6 -great omentum 15 50 dead larvae (Rabbit)

08 Basal part of 20 50 live larvae mesentry and • 09 .great omentum 20 50 dead larvae 0 10 'Basal part of 20 50 live larvael 1. Extract of larvae mesentry and - intravenously 011 ,great omentum 20 50 dead larvaej injected three times. 2. Treated 21 days after the first injection. 1 57

In the above experimental results, it was observed that some of the larvae with heat treatment became necrotic in the stomach wall after 7 days, and in 15 days they were completely necrotized. Moreover, in the results of burying larvae after injection of a larval extract, abscesses accompanied by strong eosinophilic infiltration formed around the necrotized'larval bodies or the cuticle after seven days. When larvae were buried at two times, the histological reaction was the infiltration of cells composed of neutrocytes and even if larvae were buried seven days after 30 live larvae were administered orally, nothing but the infiltration of cells such as neutrocytes was observed, and the formation of granulomas in the stomach walls of the dogs was not observed. With regard to the intestinal wall of the dogs, the histological reaction after seven days was stronger than in the stomach when larvae with heat treatment were buried, and when larvae were buried after the injection of larval extract the reaction around the larval body was marked. In the experimental results of rabbits which were different from those in dogs, •seven days after the single burial of a larva marked eosinophilic infiltration had already formed around the larval body, and after ten days 'an eosinophilic necrosis picture had appeared and in 20 days the state wàs almost identical to that in human cases. 158

After 30 days the larval body remnants had disappeared, and after 50 days the body was completely absorbed. In rabbits, the histological reaction was weakest when the burying was performed only once. In the burying experiment following steroid injection, the local histological reaction tended to be restrained. 50 live larvae were left in the abdominal cavity and 7 days later more than half of the larvae were recovered alive, but after 15 days all the recovered larvae were dead. It was clarified from the above results that there is a participation of a local allergy in the pathogenesis of anisakiasis. However, there are differences in local reaction between dogs and rabbits even if the same substances and methods are used, and it was found that changes similar to human cases can be brought about in rabbits, but it is difficult to do so in dogs. . Kikuchi et al. (1967) observed intestinal anisakiasis macroscOpically and histologically by using rabbits in which live Anisakis larvae and the partial antigen of the larvae had been injected intracutaneously, from the experimental, immunological and pathological standpoints. The results were: 1. The appearance of the first-time infection by Anisakis larvae in the hypodermis of .rabbits was one of a foreign body reaction and the second-time infection picture was one of an allergic reaction. 159

2. The helminthic allergic reaction is strongest on the second day in the case of live larvae. 3. In the subcutaneous reaction to the partigen of live larvae, for the supernatant liquid, strong edematization and reddening were observed from the 4th hour and the reaction was at its maximum between 18 and 20 hours, and for the precipitate the reaction was slower. 4. The histological picture at the time of strongest reaction was quite similar to that of human intestinal anisakiasis around 24 hours after its onset. 5. The acute intestinal anisakiasis accompanied by strong edematization in human cases is caused by an allergic reaction due to the frequent migration of live Anisakis larvae into the intestinal wall.

moue et al. (1968) also investigated the experimental' granulomas produced by Anisakis larvae in rats and guinea pigs and compared them with human cases, and found that there are differences in the host-side reactions of different 65 animals. At the time of stomach penetration, a strong eosinophilic phlegmon picture is shown only in the stomach submucosa in rats; but in guinea pigs, marked changes accompanied by strong edema and haemorrhages also appear in the tunica muscularis and serosa. 160

On the .assumption that there might be a possibility of establishing sensitization without the condition of infection due to Anisakis larvae invasion, Ishikura.et al. (1968) and Ishikura (1969) carried out the oral administration of lyophilized powder of approximately 20 larvae every day, and they found that in only one out of ten rabbits and ten guinea pigs, the antibody value was elevated four times according to the superposition precipation method, and the intracutaneous reaction was positive with a 14 mm variation in the swelling. Then, live'larvae were buried in the ileum subserosa of the rabbit and three days later the local region was resected for microscopic examination. As a result, edematization in the intestinal submucosa and allergic reactions such as a heavy eosinophilic infiltration around the larval body, and a larval body necrotization picture which was accelerated compared with the first-time infection experiments, were dbserved. Moreover, they assumed that the secreted material and excrements of larvae and the remnants of dead larvae are absorbed as antigens and hosts can be sensitized by them • Furthermore, Ishikura (1968, 1969) sensitized rabbits by using four sorts of antigen, i.e. Anisakis larvae, protein albumin, cou i bacillus and dog asdarids, and carried out detailed pathologic histological investigations on the localized enteritis of the rabbits caused by the 161

injection of the same or different antigens in the ileum subserosa. In this experiment the localized enteritis due to buried Anisakis larvae or injection of Anisakis larvae

. antigen in the subserosa presented the same histological picture as in human acute localized enteritis cases. Therefore, he deduced from these animal experiments that intestinal anisakiasis is a secondary allergic change due to re-infection, whereas gastric anisakiasis is a reaction change due to a first infection.

Mainly Concerning the Conditions of Larval Migration into Host Bodies (Distribution and Detection of Larvae)

Judging from the above infection experiments with mammals such as dogs and rabbits, Hirao and Yamaguchi (1964) . helà the view that these animals are not suitable hosts for Inisakis larvae, and they attempted infection experiments in birds (domestic fowl), amphibians (bullfrog) and fish (silver carp). First of all, oral administration was undertaken with silver carp, but the'larvae were necrotized by the time they entered the stomaCh. Then a small incision ' was performed in the abdominal wall and the larvae were imserted through it. Most of them were observed to be still 162

alive in the abdominal cavity after 40 days. Larvae inserted into the abdominal cavity of chickens one month after hatching were alive up to the 5th day, but from the 7th day they were all dead. In the case of chickens, the life span of larvae was shorter than in dogs or rabbits. In bullfrogs, the larvae had already penetrated through the stomach wall and migrated into the abdominal cavity three hours after infection by oral administration, and haemorrhages were observed in the stomach wall after two days, and five days after some of the larvae were forming cysts surrounding their coiled bodies. In observation after 30 dayS, most of them were still alive and when these larvae were administered orally to other bullfrogs, it was observed that they again penetrated through the stomach wall and migrated to the abdominal cavity. Yokokawa et al. (1965) carried out oral administration of 9 to 14 Anisakis-like larvae obtained from Scomber japonicus to rats, and they observed the conditions of larval migration into the host body and the lesions caused by the migration at various times between 18 hours and 40 days after the infection. In the observations from 18 hours to 4 days, many of the detected larvae were boring deeply into the inner surface of the stomach wall, some others were penetrat- , ing through the intestinal wall and some had already migrateà to the abdominal cavity and were boring into the great 163

omentum, lesser omentum or inner surface of the abdominal wall. Between 6 and 30 days, the number of larvae detected decreased and they no longer existed in the stomach wall but were boring into the soft tissues such as the great or lesser omentum, mesentry tissues or tissues around the internal organs of the abdominal cavity, and larvae in the process of necrotization were found in the localized granulomas. After 40 days no larvae were detected and the lesions were not clear, but in a second infection experiment there was a tendency for the histological reaction to be increased around areas penetrated by larvae or in the granulomas, and the eosinophil or plasma cell reaction was conspicuous. Isobe (1965) gave Anisakis larvae which had been parasitic on marine fish to three dogs from April 1964 to March 1965. The first dog died 126 days after consumption of the larvae. Two male larvae were found in the small intestine and two female larvae were found in each of the small intestine and stomach cavity. The second dog which received 30 larvae died 112 days after consumption and a tumour was being formed in the stomach wall. The third dog . which was given 37 larvae was killed 159 days after the administration and an autopsy was performed, but no larvae « were found. Yoshimura (1966) carried out oral administration 164

of about ten Anisakis-like larvae collected from Pneumatophorus laponicus to rats, and he observed the state of larval migration and the host-side lesions (Table IV-12).* As a result, 44 hours after the infection it was observed that some of the larvae administered had obviously already penetrated into the stomach wall, and a few had migrated to the abdominal cavity . and were boring into the caecum or the internal surface of the abdominal wall. After four days, no larvae were found in the stomach wall, and some, although only a few, of the larvae which had migrated to the abdominal cavity were boring into the soft tissues such - as the lesser or great omentum or into the adipose tissue around the internal organs and they were in the process of being gradually covered by fibrous tissues. On the other hand, in an experiment in which young dogs were given the same larvae, the larvae were observed to have penetrated into the stomach wall or pigrated to the abdominal cavity in two dogs . out of six. Table IV-13* shows the results of the above experiment, and the two dogs in which larval penetration into tissues or migration were observed had both been infected repeatedly. The histological or parasitological

. states in the penetrated area of.the stomach wall Were'quite similar to those.in human cases, and they coUld be considered . a$ a reproduction of human Anisakis-like larva migrans.

* Translator's footnote: Tables IV-12 and IV-13 appear to be in the wrong order. 1 65

I.• :g1 e, ,, - :... r., : 7 , cr. / 1-3 0 17 9 a (i) ( fîot:i L ) 44 e 2 2. — 4 . ' 12 Fi ''..1 30

• 41 el 3. '.. v... :::. 0 178M . 68. ne - -.. r., 42 4 3 . 0 n() .2.v..4? 42 .›. ..„. ; 2is h ■r- 9U 1 42 9 8 0

Table IV-12. Experiment on Infection of young dogs with Anisakis-like larvae (from Pneumatophorus japonicus) 1. Dog number. 2. Sex. 3. Weight (kg). 4. Number of larvae administered (items). 5. Time administered. 6. Time from last administration to autopsy. 7. State of autopsy. 8. Number of larvae detected. 9. Parts. 10. Remarks. 11. For 12 days. 12. For 17 days. 13. For 28 days. 14. For 28 days. 15. For 28 days. 16. Days. 17. Stomach. 18. Abdominal cavity. 19. Natural death (no food). 20. Killed. 21. Natural death.

166

a (1>r , Jet,• - -,fek ra- Q ® ,g) ID e-9-e effie g ±U ue usl

1 (WgF1)@ 113M- M 9 6 4 1

7 3 3 22114 NP 9 3 1 2 (Wi-,i- Ph) iik ,2.1e.‘ 9 2 1

44Mle 9 4 3 1

4 8 9 1 1

• se . 10 • 5 4 1

88 10 1 I l " /

9 0 11 13 10 0

128 10 1 . I

170 9 I 1

2313 14 3 1 •

• 2 • 3013 1 •

9 0 408 9 0 •

..1 k.,p4U3 C) Table IV-13. Larvae detection conditions in an experiment on infection of rats with Anisakis-like larvae. 1. Time. 2. Number of larvae administered. 3. Number of larvae detected. ' 4. Stomach. 5. Small intestine. 6. Caecum. 7. Abdominal cavity. 8. Lesser omentum. 9. Great omentum. 10. Others. 11. Hours. 12. Days. 13. (Inside the intestinal canal). 14. Abdominal wall). . 15. Epididymis). . 16. Alive ?). . 17. (Dead ?). 18. * Degenerated, necrotized larval body. 167

Okumura (1967) undertook oral administration of ten larvae doses to rats, each ten being collected from one of the fish Pneumatophorus japohicus, Trachurus japonicus, Dentex tumifrons, Trichiurus lepturus, or Ommastrephes sloani pacificus, and performed autopsies at various times and investigated the infection conditions. First of all, looking at the experimental results of the administration of larvae infecting Trichiurus lepturus, Theragra chalcogramma and Ommastrephes sloani pacificus to rats (Tables IV-14 to IV-16), the infection behaviour of larvae from these different fish species was very similar, and it was observed that between one and four days after infection larvae had bored into the stomach wall and the abdominal cavity organs, and subsequently the number of larvae boring into the stomach wall decreased as time passed, . but larvae stayed for a long time inside the various abdominal cavity organs or ihside the musculature. The - number of penetrating larvae decreased in the order great omentum, fat-body, mesentry, spleen and testicle. The percentage of larvae borihg into the great omentum was 33% of the total number of larvae penetrating into the abdominal cavity organs and musculature. 'Thus, it was found that some of the larvae penetrate the stomach wall, migrate to the abdominal cavity and bore into the various abdominal cavity organs or musculature within 24 hours of administration.

168

, ryr f Ret I35: 011"5..-A 5 (,..11, era-223 •L""frAi)

Table 1V-14. Results of larvae administering experiments using larvae parasitic in Trichiurus lepturus.

1. Number of days passed after administration. 2. Number of larvae detected. 3. Detection percentage. 4. Parts where larvae were detected. 5. Stomach. • 6. Small intestine. 7. Caecum. 8. Large intestine. 9. Great omentum. 10. Musculature, Adipose tissues. 11. Mesentry. . 12. Testicle, Ovary. 13. Prostate. 14. Pancreas, Liver, Spleen. 15. Abenteric wall. 16. Vas deferens. 17. Bladder, Uterus. 18. Numerals show the number of larvae, which penetrated into tissues, and Circled numerals show the number Of larvae in the free state. Ten larvae were administered per rat. (These notes also apply to Tables up to 1V-22.)

169

04 ue. '4 c-, e 11 e 1.i- ii 41 t AAA. ,. bi3rz tn 0 C) (7) 0 0 r, • . i tti.t. it • #4 104 ›.1e mil A . „ i V'All - 14 ‘4. • " ee oliW ;re U.. Z re tei w 2 I 40% 1 1 6 4 (D I I 1 2 4240 _ 5 4W 3 30 I a.) 2 2 4 25 3 1 1 1 3 ID 1 T. 2 (D 1 3 1 I 1 6 20 1 1 0 7 5 I 1 0 8. 4 2 I 1 0 9 0 0 . 1 10 10 1 1 1 6 20 30 4 1 1 0 0 30 0

Table IV-15. Results of larvae administering experiments • using larvae parasitic in Theragra chalcogramma Key as for Table IV-14.

0 a q) it tet t8...• e til. lk -5 a lit ilt le .1q. WM mis A Anhq.3 3èreg eiçii . Call %" hi.51-t1 le lei 1.4:.is't .11 a 4DGeent e i 2504 2 3 3 3 1 1 35 2 1 1 . I 1 3 20 3 3 . 7 2 2 3 4 45 2 I I I 5 15 2 1 2 1 I 620 2 1 1 5 I :D 1 2 7 30 1 1 3 2 1 8 15 0 0 9 5 I • 1 0 . 10 0 ' 0 0 2°. 0° 0 ' 30 0

Table IV-16. Results of larvae administering experiments using 1arvae parasitic in Ommastrephes sloani pacificus.

Key as for Table IV-14. 170

Next from the infection behaviour of larvae collected from Pneumatophorus japonicus, Trachurus japonicus and

Dentex tumifrons in rats at various times (Tables IV-17 to IV-22), it was observed that for larvae which were parasitic in Prieumatophorus japonicus the percentage of larvae pene-

trating into the stomach wall was high up to 20 hours after the administration and this penetration was observed until

30 days 'after infection, but there were few which penetrated through the stomach wall, migrated to the abdominal cavity and bored into the abdominal cavity organs or musculature. In the case of larvae parasitic in 111.2:211arm_jimplap_m, they bored into the stomach wall with a high percentage up

to 20 hours after administration, and after one day the number of larvae which migrated to the abdominal cavity after penetrating the stomach wall increased. The larvae

boring into the abdominal cavity organs*or musculature were

observed at a very high percentage up to 30 days afterwards. In larvae - parasitic in Dentex tumifrons, the percentage of

larvae . detected out of the number of larvae administered

was 26% within 20 hours of administration, but most of them were observed in the free state without penetrating into tissues, so that the infection percentage was very low and the percentage of larvae penetrating into the stomach wall

, was only 1.4% of the number of larvae detected. However, during the time between two hours and two days, the number

171

of larvae penetrating into the intestinal canal wall is far more than compared with the former two cases, the percentage being from about 9% (within 20 hours) to 21% (after 1 to 20 days) of the number of larvae detected.

. D (k) Q. (D e i* * 1 Re lei 11k4.Are, 5.1M 4vd-', 0 ® G■ (t. Et, xi . t. it* Se • 11.f ' ,. Agnk • et ,lqg eel' :id% ..-.1111 Ar, em We. 1.1i 55m, ea* . tor*. .i...-A• -0 0 0 e 9 2 (1) 12D T 10 3 (2) ■Se 2 10 00% 4 CD (i) 8 40 0 24 '2) il i 1 3 30 5 cD CD 2. (1) CD 5 3 0 2 (D 4 2 ' (D. 1 10 1 20 • (1) 1 I l I 4 3 (D 4 4 . 15 5 34 3 (D 1 2

' 4 1 (2) 1 30 • . 7 1 (41 1 1 0 •

Table 1V-17. Results of larvae administering experiments using larvae parasitic in Pneumatophorus japonicus (within 20 hours). 1. Number of hours elapsed since administration. Remainder of key as for Table 1V-14.

1 72

0 CD p le lk te vie Be en te'e %..) rite 0 0 Ill MI Jtos In ' /Val est. ,nati t",' . ff 16 -51`k elilif i_"-à-'• .Isms A5 A5 ?3.II1 ' • ia et, , 0 el, W 4,, qi r,..1 W 0 1 3 20.0%2 1 3 3 3 2Q 2 4 33.3 3 1 3 ' 3 1 1 - 3 0 13.3 . 3 1 1 CD 0 4 2 13.3 1 1 2 I . 1 0 5 1 3.3 1 ' 0 • 1 1 6 0 3.3 0 . 0 7 0 3.3 I 1 o 8 o o o o 9 o 0 o o 10 1 3.3 1 0 0 20 1 3.3 1 • 0 1 1 30 1 10.0 1 1 1

Table IV-18. Results of larvae administering experiments using larvae parasitic in Pneumatophorus japonicus Key as for Table IV-14. 173

CD 0 t te sl .,...« , 1 a),4:...:, . q) m at 15 fli 11...:1 .•;.1 .4 .:-.." %) co iz, ‹!i r'‘. 11 •1•11 'î1.4 A ! Alit Um" Al•re,4 5 11.., . c,. a t.e '.4 '„" g“t,51 reort 'î.n.,Ï.t

4 3CD . 5 3S

4 2 0 2 I (D 3 3 1 CD 5 0 16

2 2 5 2 2 3 2 10 2 34 1 1 • 5 1 2 1 1 2 1 1 2 1 1 • 2 . 2 • 15 2 28 2 5 1 1 1 1 3 2 0 5 3 1 (1) 2 0 I 20 5 40 3 2 4 3 1 4 4

Table IV-19. Results of larvae administering experiments lising larvae parasitic in Trachurus japonicus (within 20 hours). 1. Number of hours elapsed since administration. Remainder of key as for Table IV-14.

174

0 0 (3) * 1 e a 19 4 i& thso re ere e"14 (11 0) CD M • ; se • mi• mest • lit +Ai ems çmr ziliis ms_rems 99 !!`. 1 11'.1 • mumul. seme +— II 2 2 1 0 10.0% 1 1 0 . 2 5 • 16.7 Q) 1 .2 1 0 0 3 0 13.3 4 3 1 1 (1) 4 113.3 • I 2 10 4 0 0 10 5 1 20.0 1 'I 2 i I 6 2 13.3 1 1 0 3. 3 7 116.7 I • I 1 0 . 8 310.9 1 2 . Ci 0 9 0 10.0 3 0 2 3 2 I 10 2 26.7 1 1 ' 3 1 CD (D o 20 216.7 1 1 2 1 _ I 4 2 1 I 30 2 30•0 I 1 3 2 0

Table IV-20. Results of larvae administering experiments using larvae parasitic in Trac_Lhürus_imonlçus Key as for Table IV-14.

175

0 0 p -Q)e it ,fe t g fl ..' 5. . ■ ,4 ,,. , ,,

lk •A w 4; m* ;PA .,, nr.nk e let.. • ps.,IttRU. i'e ff Mtn!, 4;W/ î..e.tt •

9 0 ED -(e qbm nez5 iipoe 8 0 0 0 2 3 62% 0 9 - 2 0 0 2 0 1 3 (Dl 0 5 4 26 la) CD 0 3 CD 1 (D I I . 1 (D , s 0 1 0 10 I 20 0 o 3 CD 0 1 CD • 3 0 15 2 16 a) a) 2 • a) a) o . 2 CD (D i iii 20 I. 14 • 0 2 1 CD - 1 CD •

Table 1V-21. Results of larvae administering experiments using larvae parasitic in Dentex tumifrons (within 20 hours). 1. Number of hours elapsed since administration. Remainder of key. as for Table 1V-14.

176

'(:). ID Q % * i a, t* (r- m -5 111 Mt e±:v smrie eri 6) 1r •X emi jiii AI M• immix it, • !mto.% w • mi t“,.2 _ mg e te • (fDl&O Cle CD 0 2 I . 1 1 1 16.7% 1 2 2 • - 2 1 1 2 0 13.3 2 1 1

3 1 6.7 I 0 . .0 4 0 0 . 0 0 5 2 10.0 10 I (1) 0 .

0 0 . 7 0 0 0 0

0 0 9 0 0 0 0 10 0 0 0 0 20 0 0 0 0 30 0 0 0

Table IV-22. Results of larvae administering experiments using larvae parasitic in Dentex tumifrons

Key as for Table IV-14. . 177

When the infection behaviour of larvae, collected from these three different fish species, at different time intervals after administration is summarized and compared 0 , (Table IV-23, Pis. IV-1,2), the larvae parasitic in Pneume ,Is'ap.....o/112Lis can be distinguished and classified as stomach wall penetrating type; larvae parasitic in Trachurus japonicus as stomach wall and abdominal cavity organ penetrating type; and larvae parasitic in Dentex tumifrons as intestinal wall penetrating type. The migration routes of these larvae are shown in the schematic diagram

of Fig. 3. When Okumura's results are summarized, the following things are ascertained. The larvae from any of the fish species used have infectivity towards rats. As a general rule, they follow the route of entering the stomach wall, penetrating through the latter and boring into various abdominal cavity organs and the musculature. During that time no affinity for organs can be observed. The larvae detection percentage in rats decreases as time elapses. There are definite differences - in infection behaviour among larvae collected from the different fish species Pneumatophorus japonicus, Trachurus japonicus and Dentex tumifrons, the larvae from Dentex tumifrons being unique with a low

. infection percentage.

178

w 14 .-." 14 uuri4)!an v (73) â ia 41Q1 rf 4 '.• re. el' 11 le 4 le .,: ift 14'). Mis q.À. hl 141 3 66 0 9 0 9'2 0 (!il 1 Cl 11 "."-In- 79 87.7 3.8 83.8 iii4 *I,' 90 11.4 1:3 5.1 94.9 53 12 0 0 0 0 3 0 15 65 .3:.‘-? 11 1 . 13.8 66.3 1.3 15.0 3.8 18.8 81.2 . 1.5 9 1 55 2 26 0 0 0 0 0 0 3 81 4D1 83 84 93.3 1.2 65.5 2.4 31.0 3.6 96.4 25„.,.., ',..,. 39 24 1 25 0 0 0 5 b 0 46 54 , 250 ''' '''') 39.0 24.0 1.0 25.0 5.0 6.0 46.0 54.0

2- 25 43 9 1 3 0 0 0 0 20 4 64 16

•■ ,z ) 250 60 326 . 53.8 11.3 1.3 3.8 25.0 5.0 80.0 20.0

51 22 2 15 1 1 2 23 1 1 7 62 eri 1 4 69 26.8 1.4 31.9 2.9 21.7 1.4 1 4 2.9 33.3 1.4 1.4 10.1 89.9 0 0 0 0 0 0 12 2 30 2 ee.e 36 32 8.9 18 0 56.3 37.5 6.3 93.8 6.2

0 0 0 0 0 45 8 48 10 b 1--308 7 , 36 58 16.1 3 2 0 ê "; 560 5.2 3.4 77.6 13.8 82.8 17.2

0 1 0 2 0 7 2 11 3 ,.e e 4 V, ,,, 391 1 1 0 " . 34,9 - - 7.1 7.1 7.1 14.3 50.0 14.3 78.6 21.4 Cr>i.ek11:1,,TT%ueLkmu:71t. -cco

Table IV-23. Larvae administering experiments using larvae from three different fish species (comparison at different time intervals). 1. Time Passed after administration. 2. Fish species. 3. Number of cases. 4. Number of larvae administered. 5. Parts. 6. Number of larvae detected. ' 7. Actual number. 8. Stomach. 9. Penetrating. 10. Free state. 11. Small intestine. 12. Caecum. 13. Large intestine. 14. Abdominal cavity, Organs. 15. Total penetrating larvae. 16. Free state ones separated. 17. 30 minutes to 1.5 hours. 18. 2 to 20 hours. 19. 1 to 30 days. 20. Pneumatophorus japonicus 21. Trachurus japonicus 22. Dentex tumifrons 23. The percentages shown under 'Penetrating"Free state' are percentages of the number of larvae detected.

179

EF) 0 - À I I --- .3i#11

C) a , u5, --Œ7) .R1t 0 e.

- - • L

cg) 4

0 4 ' ' 70 50 ' 7;0 10 0 10 30 00%

Fig. IV-1. Behaviour of larvae detected in rat bodies (within 20 hours of administration). 1. Pneumatophorus japonicus 2. Trachurus japonicus 3. Dentex tumifrons 4. Detection percentage. 5. In the alimentary canal or on the alimentary canal wall. 6. Penetrating through. 7. Abdominal cavity. 8. Penetrating. 9. Free state.

ffl — À

;ei ei• C) x

0 *9.4

e) aa loo 70 50 30 10 0 10 50 70 100

Fig. IV-2. Behaviour of larvae detected in rat bodies • ( 1 to 30 days after administration). Key as for Fig. IV-1. 180

o etÂ

Mk e F1

ffrrnmi 7 a7 NUM (D)ÇM1111. 1.et.een .i ;T-s -r

Fig. IV-3. Proposed behaviour of larvae from three fish species in rats. 1. Larvae. 2. Penetrating. 3. Stomach. 4. Stomach wall. 5. Penetrating through. 6. Inside abdominal cavity. 7. Sojourn. 8. Intestinal wall. 9. Organs, Musculature. 10. Intestine. 11. Sooner or later, they are to be absorbed into tissues and destroyed or degenerated as foreign bodies. 12. Pneumatophorus japonicus 13. Trachurus japonicus 14. Dentex tumifrons 15. Heavy line shows the main route. 181

Matsuoka and Usuya (1966) infected dogs with larvae obtained from Thunnus thynnus, Scombrops boops, Scomberomorus /11.029121.21, Katsuwonus pelamis, Lophius litulon and Ommastrephes sloani pacificus, and performed autopsies one to three days after the infection. The larvae were all boring into the stomach wall mucosa with the anterior part of their body, and some of the larvae from Ommastrephes sloani pacificus were found not only in the stomach wall but also in the small intestine wall or in the abdominal cavity, and it was ascertained that all these larvae infect dogs easily. Moreover, when larvae obtained from small Trachurus japonicus had their posterior end half or quarter amputated and then .were administered to four dogs, small haeMorrhagic spots were observed in some areas of the stomach mucosa two or three hours after *administration, but no laryae were seen to have penetrated with the anterior part of their body. Therefore,. it was decided that infection by amputated larvae cannot occur. Nagase (1968) investigated the conditions of'migration of.Anisakis type I larvae.into the bodies of rats. As-a result, the penetration of larvae into the stomach wall began one h6ur after the oraladministration,..and most.of the larvae penetrate or pierce through the stomach . wall after about eight hours. The larvae infectivity is higher when hosts are starving than when they are full.. There was ( • 182

no remarkable influence on the penetration of the Anisakis larvae into the stomach wall even if the rats were treated beforehand with medicine which acts on the syMpathetic or parasympathetic system or crushed larvae liquid was administered orally or injected into the abdominal cavity of the rats. Therefore, it was presumed that there is more weight in the larvae-side preference for invading the stomach wall rather than in the above host-side conditions. Shiraki (1969) undertook oral administration of 73 Anisakis types I and II, Terranova and Contracaecum types (A) and (B) larvae to rabbits and dogs, and investigated the fate of the larvae by performing autopsies 3 to 24 hours later (Table IV-24). Those which entered the stomach wall were.Anisakis type I and Terranova larvae, but there was no sign of Anisakis type II penetrating although most of them were recovered alive six hours after administration. With Contracaecum type (A) larvae, all larvae recovered after three hours had lost their motility, and type (B) larvae were digested and many were broken into pieces, and there was no sign of penetration in either type. 74

183

0 IV.5. (>) tt in L5i7 tu to, W. o 0 'it- - - +4 0 1 C) rÈ ) * 1 a tt 93 14 a (2) * +I) 10 VP* A ese ez ( DI NI ) e

Q 0 0 :9' V '67 Anisakin I () 7. 15 /2 -5 6 I, eelep . 61.

X iegi 10 11/ 1 '67 Anisakla I 7 @$ ,' 30 '2 » 24 1 +MO 1 AII.F1C) 2

•% ,,,,,D i { Aninakis I riel:, 1- 12 ., Haw," 4/V '67 1 y ,,, 6 (94 I .. la c) 9 , ,t, 3 2 I 6 Aniaakis II ,,C),., e 3 '`<[:■ 3 0 . 19/ V '67 Anisakia II 43.4 3- 30 .7 11 91 3 rte 5 .1. tiaâ1

I WEtl 3 19/V '67 4h ■ ••41. 11 nalf 4 25 ' 't Y 2 i lo ' EP s 4 3 i 1 ,4•0 6 2

6. »71 • '6 7 Terranova 7'9,1 4, I 4 1 .. %. 6 ve @) 4

5/ X :67 'rerranova vq...» ' 3 •/ .1. Y 1 51 1D 3 6/X '67 Terranova w(j)4 e r 24 a .,..:, , ii,.. .67 Terranova ?el .. 5 5 .., 1 9' 3 ( 11.1`; I

OR P , 11.VCW11 ; I. i 43 ,...... '3 2. 12/X '67 Terranova 6 1 ;:', 3 1 ,! 3 .3(à) 1 ; eaGee .2. ' e. 'S e 8 .) 1 4 7 27/Xl '67 Terranova , e). ,• 9 '7 7 •.‘• 3 er .1 Z V® 0 I 17 la is I 17/X( '67 Cc:Ur/tramcar.% A ›.WY, SO ^,• • 4 3 '-' 14 3 1 4 3 19/VI '67 Contr•ea•eum 13 7.ti:.1( -I r/ 15 ^7 e 3 WO 8 5

23/VI '67 Co/Ur/matron 11 7.3f h 20 ^tig'le. 3 I lit eI

Table 1V-24. Anisakis larvae infection experiment (Oral administration carFied out January 1967 to December 1967). 1. Date. 2. Species of larvae. 3. Host. 4. Number of larvae administered. 5. Experimental animals. 6. Administration - Autopsy (hours). 7. Recovery condition of larvae. 8. Penetrating larvae. 9. Non-penetrating larvae. 10. Live larvae. 11. Dead larvae. 12. Complete. 13. Damaged. 14. Pieces. 15. PL122.111m9.11m_ilm211 16. Phiiiiii.iii-37aib-onicus 17. Katsuwonus pelamis 18. Hippoglossoides dubius 19. Gadus macrocephalus 20. Stichaeus grigoriewi, Sebastolobus macrochir. 184

21. Érctoscopus japonicus 22. Theragra chalcogramma 23. Ommastrephes sloani pacificus 24. Rabbit. 25. Adult dog. 26. Stomach wall. 27. Abdominal cavity. 28. Lesser omentum. 29. (9 haemorrhagic spots in the stomach mucosa). 30. - (3 haemorrhagic spots in the stomach mucosa and subserosa). 31. Stomach. 32. Small intestine. 33. Caecum. 34. Colon.

Mainly Concerning Factors of Both the Host and Parasite Sides

Asami et al. (1964, 1965) carried out oral admin- . istration of Anisakis 'larvae of 2 to 3 cm body length collected from Pneumatophorus japonicus to dogs, and out of six dogs he found one which was given 45 larvae daily. for nine days to be infected. .Larvae were found nowhere in the alimentary canal apart from in the stomach. Moreover, Asami et al. (1965) collected Anisakis-like larvae from the abdominal cavity, visceral organs and abdominal musculature of Pneumatophorus.japonicus l and undertook oral administration of these larvae to guinea pigs and investigated the presence and migration conditions of the larvae by pérforming autopsies after certain times. Establishment of infectiOn occurred in 4 out of 20 untreated guinea pigs, 185

and out of 15 guinea pigs which were given an autonomic blocking agent, 11 were actually infected. The places where larvae were found were the mucosa of th'e stomach and samll intestine, colon, caecum mucosa, abdominal cavity, adipose tissue of the liver in the abdominal cavity, etc.. He reported, moreover, that the freshness of the larvae influences whether the infection occurs or not. Similarly, Asami (1966) and Inoshita and Asami (1967) investigated the factors connected with the establishment of anisakiasis infection. That is, they collected larval . 75 nematodes with an Anisakis-type oesophageal structure from the abdominal cavities of Pneumatophorus japonicus and Theragra chalcogramma, and administered them orally to guinea pigs which had been given various treatments and then investigated the infecting larvae by performing autopsies after certain times. When guinea pigs which had had a hyaluronidase preparation inserted intb the stomach 30 minutes prior to administration of the larvae for the purpose of destroying the tunica of the larvae were compared with untreated ones, no difference was observed. With guinea pigs in which the same preparation was injected intramuscularly, a slight increase of the infection percentage was observed compared with that Of the'control group. The same result was obtained , when guinea pigs were given larvae after having daily intra- muscular injections of 5 mg of adrenal cortical hormone 186

(cortisone) for three days. Next, when guinea pigs which were re-infected 24 hours after the first infection were compared With those which were infected only once, the number of larvae was less in the re-infected ones . than in the others. Therefore, as there seemed tc be a preventive effect due to the first infection, they tried re-infection after 48 hours and 10 days, but the results were exactly the same as those of a single infection. Therefore it was prèsumed that there was no re-infection preventing mechanism. The following experiments were also carried out by Asami (1966). Asa parasite-side factor, the infection percentage was distinctly high with medium-sized larvae of length between 2.3 and 2.5 cm. In an experiment in which the.anterior parts of amputated larvae were used, there was still an infectivity even if the larvae were cut into half. As a host-side factor, in an experiment on sensitivity in guinea pigs of different weight the infection was highest . for weights around 500 grams. Moreover, the infectivity was slightly higher in starving guinea pigs than in those having a regular diet. Concerning Anisakis infection, the infecting factors of the larvae are stronger than the host- aide conditions, and when,the infecting ability of the . larvae is decreased for some reason, then the host-side conditions become effective. Thus he concluded. 187

Mainly Concerning the Clinical State of Animals under Anisakiasis Infection

Matsuoka (1965, 1966) divided 35 rabbits into six groups and carried out.oral administration of larvae only once for the first group; repeated administration for the second group; for the third group larvae treated with hot water were buried in the stomach wall; for the fourth group the larvae were orally administered or buried in the stomach wall after the injection of hyaluronidase, antihistaminics or steroids; for the fifth group live larvae or larvae given heat treatment were inserted into the abdominal cavity; for the sixth group an intravenous injection of crushed laryal body liquid was carried out three times at two-day intervals, then two weeks later live or dead larvae were inserted into the abdominal cavity or administered orally or buried. As a result, there were no marked changes in the number of red blood-corpuscles and the amount of hemoglobin among any group, and the white blood-corpuscle count was increased teMporarily until 12 hours after the oral administration, but it did not increase further after that. Regarding.eosinocytes, their number increased temporarily in both the single and repeated oral administration groups, but in the groups which were treated with medicine or for 188

which the buried larvae were treated with hot water, their number did not increase. However, their number increased between 4 and 15 days after administration in the group in which live larvae were• inserted into the abdominal cavity and in the. group injected with the larval body extract. There was no marked change in the amount of serum protein throughout the whole time, but the .amount of albumin tended •to decrease slightly and the Y-globulin increased a little. In:experiments on administering Anisakis larvae to rabbits, • the larvae generally penetrate under the stomach submucosa or subserosa with single oral administrations, and with the stomach repeated oral administration they invade not only but also the small intestine, caecum.and.large intestine. .From . a histological vieWpoint, a marked eoàinophilic granuloma is formed around the larval body or the necrotized body, and pseudo-eosinocytes increased remarkably up to 24 hours • after administration and the eosinocytes were at a maximum between 10 and 15 . days after the administration.

Concerning Examples of Experiments in Other Countries

As an example of research in other countries, Myers (1963) carried out oral administration of Anisakis-type • 189

larvae to 36 guinea pigs, which were killed at eight-hour Intervals after infection. The larvae were found in the stomach, liver, mesentries, pancreas, small intestine, large intestine, caecum, thyroid gland, fatty tissue around the kidneys, and in two guinea pigs larvae were observed encysted under the skin. Living larvae were observed in the bodies of the guinea pigs for up to five days; however, within six days all larvae had disappeared and no trace of tbeir presence could be observed in any part of the body. Moreover, in the autopsies of these guinea pigs, in the macroscopic state a haemorrhagic spot was observed within 24 hours at the site where the larvae had penetrated the stomach and after 48 hours it could not be seen. When Kuipers (1964) administered Anisakis larvae collected from herrings to rabbits, several larvae had penetrated the mucosal layer of the gastrointestinal wall within 24 hours, causing some slight reaction with necrosis 80 and slight eosinophilic infiltration. After three to four days, the larvae appeared to be dead and causing no more reaction than on the first day. After several weeks or months calcified larval remnants could be found . dispersed through the wall of the .gastro-intestinal tract. In this experiment, the parasitism was high in the cardiac and • pyloric regions of thé stomach and the appendix. Moreover, in the repeated oral administration of larvae to rabbits, 1 90

if two larvae of different dates of administration were found within 1 cm of each other in the gastrointestinal wall, the histological reaction was severe. However, if the larvae were administered with an interval of four months or more, no extra reaction was found. Kuipers concluded that the penetration of a larva into the wall of the gastro-intestinal tract resulted in a local hypersensitivity in a very small area which subsided after about four months, and he considered local allergy •as an important cause of the pathogenesis of anisakiasis.

pummary:

In this chapter, we introduced research on experimental anisakiasis for the purpose of investigating the cause of pathogenesis of anisakiasis infection. From the results of this research, we may deduce that the fact that the cause of pathogenesis of anisakiasis infection is a local allergic reaction restating from a sensitization in which the allergin is Anisakis larvae, their secreted substances or excrements from the larval body has almost been proved. Moreover, it . is possible to analyze the factors on the parasite or host sides in more detail. Although the relation between the 191

adult nematodes parasitic on marine mammals, for which man was originally thought to be an abnormal host, and human anisakiasis became clear recently, the question of which factors cause anisakiasis when present has remained unanswered ., However,. it is considered.that the fact that an allergic reaction is Participating between them has almost been established from the clinical, pathological and immunological investigations based on the above animal experiments. Although there will be some danger of misinterpretation if the resultà of infection experiments on ariimals are applied to human cases without modification, it is almost certain from these results that in anisakiasis at least, the Anisakis larvae as a parasite-side factor have more significance in affecting thé pathogenesis of anisakiasis infection than man as a host-side factor. . However, we cannot overlook the fact that infection behaviour .of the host side sometimes varies even if the same substance or method is used (Shinno, 1966), or that even if the same species of animal is used, some differences in infection behaviour due to the origin of the parasite can'be found (Okumura,. 1967). • It is . thought that further investigation on the infection behaviour.in different hosts and individuals as well as.detailedresearch on each larva will be necessary in the future. .

. . 192

V. FOOD HYGIENIC COUNTERMEASURES AGAINST ANISAKIS LARVAE 81

The sources of the animal protein in the diet of our country are_largely dependent on marine products. A habit of consuming raw fish as sushi or sashimi has long been established, and the taste for thià is still unchanged. The prophylaxis of anisakiasis, that is, food hygienic countermeasures, is to avoid the oral consumption of living Anisakis larvae; but as a practical matter it is almost impossible to prohibit the consumption of raw marine fish, which is the source of infection. ' If allergic reactions are considered as part of the cause of pathogenesis of anisakiasis, we must even avoid the consumption of any constituent substance of the Anisakis larval body. Although it is certain at present that the pathogen of anisakiasis is the Anisakis type I larvae, we must also consider the possibility of other closely related - nematodes becoming causes.

Resistance of Anisakis Larvae

To 'date there have not been many detailed reports on the morbific ability , of these larvae; however, the

, • 1 93

resistance of the larvae, which is thought to influence their morbific ability, has been quite well investigated in the field of infection prophylaxis. This resistance of the pathogenic organ will be the most important question when food hygienic countermeasures are considered. There are a considerable number of reports from this or other countries in which the resistance of larvae under various conditions was investigated from the viewpoint of anisakiasis prophylaxis. Especially in our country, there are systemmatic reports in this field by Yamaguchi et al. (1965) and by Kawada (1968). In this chapter, the behaviour • of larvae in different environmental conditions is .to be described, referring mainly to the results r.y Kawada. In reports on the resista.:Ice of Anisakis larvae, the life span of the larvae in various media (seasonings, salt water, acids, etc.), their resistance to high or low temperatures, the direct larvae killing effect of various medicines, etc. are generally investigated. These are explained below.

(1) Resistance in Various Media

The media used here are liquid to make the larvae

swim, and they are selected mainly from the viewpoints of 194

cookery or sterilization.

1) Salt water

According to Yasuma (1965), the life span of Anisakis larvae in relatively low concentrations of salt water (0.0 to

0.9%) was 24 days, the longest, at 0.9%, and 9 days at 0.5%. In a larvae fatality experiment in different concentrations of salt water (30%, 20%, 10%, 5%) by Kato et al. (1967), a 100% larvae killing effect was observed at 20% or 30% concentrations, a 33% lethal effect after 24 hours in 10% solution, and a 100% larvae killing effect was observed for

48 hours in any of the concentrations (Table V-1). Yamaguchi et al. (1965) and Kawada (1968) investigated the number of larvae survival days by using five larvae per each concentration of salt water from 1 to 30% at room temperature (Fig. V-1). As a result, larvae lived More than 175 days in 1% or 2% solution, but in 5% they died after 9 days, and the number of larvae survival days became shorter as the salt water concentration increased. Moreover, Anisakis larvae which were put into salt-pickled whole cuttlefish all died within 48 hours. • 195

0 E4 NI o 2 4 M re1 48M rj1 le, Z 1

30'o - 10/10 10 .' 10

20°. 10 / 10 10/10

1 0ri, 7,' io 10/ 10 5ea 7/10 10 / 10

Table V-1. Life span of Anisakis larvae in various concentrations of salt solution. 1. Concentration. 2. Time. 3. 24 hours. 4. 48 hours. 5. Remarks. 6. Number of dead larvae / Number of larvae examined.

fir 0 0 X 4 lei

5 11105=11, 175 V.I. ±.

2 5 IMUZZZIEMEM=Ci MEC21:2===i1 175 (11 5 5 me 9

10 5 Mil 7 15 5 3

20 5 • 3

25 5 $ 2

30 5 II I

15 25 155 165 175

Fig. V-1. Survival time in days of Anisakis larvae in various concentrations of salt solution. 1. Salt concentration. 2. Number of larvae at the time test was begun. 3. More than 175 days. 4. Days. 196

. Il) Hydrochloric acid

• According to the resistance to hydrochloric acid by Yamaguchi et al. (1965) and Kawada (1968), larvae lived a relatively long time between pH 0.8 (1%) and pH 5.4, except for pH 0.8 and pH 5.4 (Fig. Y-2).

III) Various acids

Also according to Kawada (1968), most larvae died in four days in 1% eulfuric acid, the longest one living for eight days as shown in Fig. V-3. In 1% lactic acid they lived a relatively long time, the longest being 30 days. In 3% carbolic acid.they all diee within ore day. In 5% acetic acid they survived a, relatively long time, the longest being 32 days. The results in vinegar were approximately the same .as in 5% acetic acid. Van Thiel et al. (1960) observed that in gastric juice - free HC1 acid 40m. eq./1, total acid 63 m. eq./1 at 37°C larvae' lived a maximum of 10 . d as, in a 2% acetic acid and 5% salt mixture at OeC the maximum survival was 25 days, and in a 1% acetic acid and 2.5% salt mixture they lived more than 25 days.

•

• 197

. .. , . . • -• li)

. , 1 0 . 0-1 *It.ft . • , ' • . ' ( 1 Ogg) 0.8 ' M 16116..... 11 .

2.0 5 MIIIMIIIMIIIIM. ffliiiiing=11 ICZCZCZIZIEVOCi 112

go ..2.4 5 IMMIIMMIRal w. _--- > Ma 90 ' 2.8 , 5 1.111111111fflaili WRIMIMMIaMa Mil 90

3.4 5 11111111111.111111 64

4.0 5 IMIIMMEZEI• 24

4.6 5 IMEEMIE! Ma 51

4.8 MN 51

5.4 5 Mara' 18 1 j 1 1 1 1 1—* 111 • • 5 .i5 .25 55 65' 95 105 .115 BO

Fig. V-2.. Survival time in days of Anisakis larvae in hydrochloric acid solutions of various pH values. 1. Number of larvae at the time test was, begun. 2. Days.

. Jigem omo)

1%1-1,SC:4 10 kit.. 8 • (p11:1 ..6) • •

9ôC.11,CH (OH )COOH 10 Willem, (pli: 2.2 )

3 %d,,H2OH IC 1 g (PH:5.4 )

5 9e•Cl1,COOH 10 ■•■ 32 (p11:2.0) iltriml

V.inegar (pH :2.0) 35

5 • 15 25 • 35 45 558 . C)

Fig. V-3. Survival time in days of Anisakis larvae in various acid solutions. 1. Number of larvae at the time test was begun. 2. Days'. 198

IV) Various alkalis

-According to Kawada (1968, Fig. V-4), the larvae could hardly live in 1% sodium hydroxide solution. In 1% potassium hydroxide most of them died within four days, and in 1% sodium carbonate most of them had died by the 18th day. In artificial intestinal juice (37°C) in which trypsin was added to 0.6% sodium bicarbonate, nine larvae died on the third day, and only one larva survived to the fourth day. Yasuma (1965) investigated the influence of various ion concentrations (M/10 erensen's buffer solution pH's 1-12) on Anisakis larvae, and found that in pH 4 they lived for 15 'days which was the longest, and in pH 12 their surviving time was one day, being the shortest. Their motility was strong in acid and weak in alkali.

V) Various nutriments

Kawada (1968, Fig. V-5) also investigated the number of days that Anisakis larvae survived in rabbit serum and in the commercially available general amino acid preparations which are used for various blood transfusion succedanea. As a result, rabbit eerum was most suitable for the survival Of Anisakis • arvae, followed by Pereston N. Es-Polytamin and 199

Moriamin S were inferior to the former two in the number of survival days. In this connection it may not be altogether out af place to add that it is probably possible to judge the nutritive value of the medicines used according to the number of days that Anisakis larvae survived. Might it not be possible to make measurements of the effects of nutriments using Anisakis larvae . -- the possibility of a bioassay ?

XUPPil ME4e) Na OH (pli :13.2) 10 I

KOH (pH :12.2) 10 h._ 6

: Na,CO, (pH: 11.4) 10 P-7:71:7à4 35

n 10 ri 4 •

( pH :10;4) 5 2-5 35 45 55l3 0

Fig. V-4. Survival time in days of Anisakis larvae in various alkali solutions. 1. Number of larvae at the time test was begun. 2. Days. 3. Artificial intestinal juice.

o el'egesee o 7 1EMOZZEMLmmummi 42 1. Number of larvae Pereston N 5 11.1.1111..m. 25 at the time test was begun. . Es-Polytamin (10%) 5 49 . 2. Days. 3. Rabbit serum.

Moriamin S 5 MIL.. g

• 5 15 25 .35 45 55B® leig..V-5. Survival time in days of Anisakis rarvae in various nutriments (blood substitute). • 200 VI) Seasonings

First of all, regarding commercially available soy sauce, the maximum survival time of larvae in soy sauce diluted to 3% was 65 days and in undiluted soy sauce they

died in one day, according to tests by Yasuma (1965). In an . . experiment by Kawada (1968, Fig. V- 6), 4 out of 5 larvae died within one day in undiluted soy sauce, the remaining larva dying on the second day. Next, concerning other commercial brown sauces, the

larvae lived for 48 days in a sauce diluted to 5% according • to a test by Yasuma (1965). In an experiment by Kawada (1968, Fig. V-6), they all died after one day in undiluted sauce. In acetic soy sauce the survival time of the larvae is generally short, 3 out of 5 larvae dying on the second day.

Some smrvived to the third day (Kawada:, 1968).

1. Number of 7.e larvae at Ø c7) . the time test was ewe begun. CD ;à 2 5 L 2. Days. 3. Soy sauce.

— 4. Brown sauce. 5 1 1 5. Acetic soy sauce (1:1). 6. Artificial gastric We II $41(1:1 5 L 3 juice. (pH: 1.4)

0 A1.11;« (p 1-1:1.41)

15 25

Fig. V-6. Survival time in days of Anisakis larvae in various seasonings.

201

In vinegar diluted to 5% the larvae survived for 51 days (Yasuma, 1965). When Kato et al. (1968, Table V-2) observed the resistance of larvae to vinegar diluted 0, 2, 3 and 4 times, there was no change in 24 hours, in 48 hours some were affected and in the usual cooking concentràtions (2 to 3 times) the survival percentage on the tenth day was around 50%. Moreover, according to Kawada (1968) the maximum survival time of larvae in pH 2.0 vinegar was 35 days. In artificial gastric juice (hydrochloric acid pepsin) at 37°C, 7 out of.8 larvae.died by the fourth day and all died by the sixth day.

laiZeo 28 4 8 68 108 W W .

• 4 0/10 0/10 3/10 10-'10 10/10 ®r. 0 /10 1 /10 1 /10 2./10 4/,10

3 3 0/10 2/10 3/10 5/10 6/10

4 • 0/10 1/10 2/10 2/10 3./10

Table V-2. Survival time in vinegar. 1. Concentrations. 2. Number of days. 3. Day (Days). 4. Remarks. 5. Number of dead larvae / Number of larvae examined. 6. Undiluted. 7. 2 times. 8. 3 times. 9. 4 times. 202

In connection with seasonings, it was r6ported recently by Kawajima and Hamajima (1966) and pthers that allyl- and phenyl-isothiocyanate, which are constituents of the grated wasabi (a kind of horseradish, Eutrema wasabi) usually accompanying sashimi, have a killing effect on Anisakis larvae. According to these reports, larvae in the above-mentioned wasabi constituents diluted to 1 / 100 became very weak in one mi,nute, and stopped moving completely in five minutes; and in a concentration of l'/ 10,000 they all stopped moving within an hour. Yoshimura and Kojima (1968) also investigated the killing effect of ten kinds of isocyanate in wasabi, and found that larvae died or stopped moving within six hours in phenyl-isothiocyanate diluted 400,000 times, allyl-isothiocyanate diluted 100,000 times, benzyl-isothiocyanate diluted 100,000 times and in (3 -phenyl- ethyl-isothigcyanate diluted 200,00 0Y -times. Kato et al. (1968) investigated the resistance of larvae to commercially available powdered wasabi diluted to a 5% or 10% concentration liquid, and reported that the larvae stopped moving in ten minutes and the lethal effect appeared after two hours. Oishi et al. (1968, unpublished), as a preparatory test, left larvae thought to be Anisakis type I collected from visceral organs of fresh Pagus major in commercial wasabi Powder for 30 minutes, then the larvae were put into isotonic sodium chloride solution where they moved actively, 203

and after leaving them in the solution for 30 minutes the larvae were transferred back to the wàsabi powder and left for 30 minutes, then they were again put into the salt • solution, but no change was found in their motility. Although this is different from the resultà of Kato et al. (1968), it is not known whether thé species of larvae were different', the freshness'of fish from which our larvae were

collected . was better, or the powdered wasabi was a different kind. •

VII) Rearin ter

According to Kawada (1968, Fig. V-7), the maximum

survival time of larvae was 75 days in Tyrode's solution at 87 room temperature, at 37°C it was shortened, being 24 days; and in Ringer's solution the maximum survival time was 62 days at room temperature and 32 days at 37°C. In city water, 9 out of 10 larvae died within 36 days at room temperature and the last larva survived for 41 days, and at 37°C they all died within 7 days. In distilled water, the longest

àurvival time at room temperature was 20 days. 204

xenn (7)

Tyrode (I PA) 5 Ir"--"--• 4ww, 75

Tyrode » (37t) 10 gewi. 24

0 _Ringer e (..ï-,z) 5 62

0 Ringer (37t) 10 32

0 *i.1* (1. 1.) 10 Mgefflelftere 41

0 ?hilt* (37't ) 10 I 7

0 M;W* (I ) 5 11113121111.».. 20

5 15 25 35 45 55 65 75 a C)

Fig. V-7. Survival time in days of Anisakis larvae in rearing liquid, city water, distilled water. 1. Number of larvae at the time test was begun. 2. Days. 3. Tyrode's solution room temperature). 4. Tyrode's solution 37 0 C). 5. Ringer's solution room temperature). 6. Ringer's solution 1 37 0 C). 7. City water (room temperature). . 8. City water (37°C). 9. Distilled water (room temperature).

VIII). Formalin

According to Kawada (1968, Fig. V-8), the larvae survived six days in 1> formalin at room temperature; in 5% formalin the longest time was 250 minutes, and in 10% fprmalin the longest was 170 minutes. 2 05

m -XeM

0,5 1 m I 4re9, 6

5 10 15 20 25 0 0

5 5 enaMMMŒrŒMMIMMEMM=M 250

10 5 ESSEIMMUMMIM 170

O 120 180 240 13

Fig. V-8. Survival time of Anisakis larvae in formalin solution. 1. Number of larvae at the time test was begun. 2. Days. 3. Minutes

• IX) 'Ethyl alcohol

According to Kawada . (1968, Fig. V-9), the larvae lived for five days in 10% alcohol, 13 days in -approximately 88 15% Japanese sake, 180 minutes in 20% and 100 minutes in

30% sake. Larvae survived for 140 minutes in 39% brandy, 25 minutes in 50%, 5 minutes in 70%, and in 100% brandy they àll died within one minute. • 206

0 74aftne- e a W40 tie.rK lc

:5 5 13

5 10 15 20 25 ( )

20 5 iffignaiênagairalliM 180 30 5 inigaiMint 100 39 5 1 40 50 525 70 55 103 51 1

120 180 240®

Fig. V-9. Survival time of Anisakis larvae in various concentrations of ethyl alcohol. 1. Number of larvae at the time test was begun. 2. Days. 3. Minutes.

(2) Resistance to Various Temperatures

Investigation of this subject is very important from the standpoint of methods of cooking or storing fish meat. In this section, we will discuss the relation between various temperatures and time required for killing and the relation to kinds of solvents and their concentrations at low temperatures. 207

I) High temperature

The principal research concerning the resistance of Anisakis larvae to high temperatures was by Yasuma (1965), Kawada (196_8) and by Van Thiel et al. (1960). Yasuma (1965) reported that the larvae died in 15 minutes at 50 0 e, 5 minutes at 60°C, and at temperatures higher than this they died instantly. According to Kawada (1968, Tables V-3,4), the survival time in warm water at 45°C was 61 to 78 minutes (average 69.1 minutes), and at 50°C they lived 7 seconds at the longest, being all dead after 8 seconds. At 60°C they_died in less than one second and at temperatures of 70°C or higher they died instantly. According to Van Thiel et al. (1960, Table V-5), when Anisakis larvae were heated in half-diluted seawater, they all survived one minilte at 45°C, 40% . of them survived one minute at 50°C and the survival percentage for 10 seconds at 55°C was 0%.

Tsty.e4 • I c. Et3f61 G 1. Temperature (°C). (t) (53) 2. Larva number. 1 71 3. Survival time (minutes). 2 62 Average survival time 3 75 4. (minutes). 4 61 5 68 69.1 45 6 78 7 64 72 9 68 10 72

Table V-3. Influence of warm water on Anisakis larvae. • 208

® m;w ft .DrJ1 fÈeke 1. Temperature (°C). 2. Number of larvàe used. 3. Time of immersion in hot water. 5 5 4 (seconds) 5 6 2 50 5 7 1 4. Number . of larvae surviving. 5 0 5. 70 or higher. 5 9. .0 6. Movement stoPped instantly. - 5 10 0 ib 1 10 2 ' 0 10 3 . 0 ep% 703-i t V 10 . t1 W1

Table V-4. Influence of hot water on Anisakis larvae.

1. Temperature. 2. Seconds. 3. Survival percentage.

Table V-5. Survival conditions of larvae with temperature.

In Low temeerature

Anisakis larvae . are generally well resistant to low temperatures. There are reports concerning this point by Yasuma (1965), Fukunaga (1966), Kagei (1967) and Kawada (1968), and by Gustafson (1953), 'Van Thiel (1960) and Vik (1964) in other countries. According to Yasuma (1965) larvae lived for 145

• 209 minutes at -20°C. According to Kagei (1967, Table V-6), larvae parasitic on fish (Trachurus jAnni.„2.121, Pneumatophorus tapeinocephalus,lamas0i -trel .cificus ) brought in on ice are all active, but those in frozen fish were all , dead. Fukunaga et al. (1966) investigated the survival time of Anisakis larvae on subjecting larvae to conditions of combinations of various concentrations of sait and various low temperatures. The results are summarized in Table V-7. AcCording to this Table, for certain combinations the life span of larvae became longer as the salt concentration became higher. This .wa.s especially remarkable at a temperature of • -15°C. Moreover, when visceral organs of Trachurus japonicus were put into a plastic bag and stored at -20°C for two hours, no larvae . were found alive.

0e.i.itkzteA4 eA tje 3f..i.t) ilii.s] . 0 .,0e 51 0,e

7 72 0 381

20 0 29 .. 30 0 22 % e 122 0 432

1 4'. .' ;* 1:11 , 14tI M 40 0

Table V-6. Survival conditions of larvae with storage methods. I. Storage methods and fish species. 2. Number of fish examined. 3. Number of larvae which died. 4. Number of larvae which survived. 5. Refrigeration. 210

6. Trachurus japonicus 7. Pneumatôphorus 8. OffiMa-Stree-S-S-16-anfTpiriC-ITS- - 9. Total. . 10. Freezing. 11. Ommasessloani_pacill

ne: ) 0 (2P71‘ 16* ) 3 6 10 15 2 50 se 30 8 M 7 5

; —10 6M MO 413 3 H 3 n 333

—15 128) rel UMM 1 6 20

—20 3BV):1 3 11 1 3 MM 2 re!

Table V-7. Survival time of Anisakis larvae in various temperatures and in various salt concentrations. 1. Salt concentrations (%). 2. 0 (city water). 3. Temperature (°C). 4. Days. 5. Hours.

Kawada (1968) made more detailed investigations. That is, he investigated the survival conditions of Anisakis larvae in each of several experiments, i.e. one in which a total of 931 Anisakis larvae were put into city water and 3, 6, 10 and 15% salt water and kept in a refrigerator at . 2°C (Table V-8); one in which 710 larvae were put in the ' same solutions as in the 2°C case but kept at a temperature of-10°O (Table V-9); one in which 550 larvae were kept 211

at -15°C (Table V-10) and one in which 500 larvae were kept at -20°C (Table V-11). Referring to Tables V-8 to V-11, when the larvae were put in city water at 2°C they still survived even after 50 days, at -10°C they lived for 6 hours, at -15°C for 4 hours and -20°C for 2 hours. However, in these experiments, their life span was longer in salt water than in city water. That is, in 3% salt water, their survival time was 4 days at -10°C, 12 hours at -15°C and 3 hours at -20°C; in 6% salt water, 3 days at -10°C, 12 hours at -15°C and 3 hours at -20°C; in 10% salt water 3 days at -10°C, 1 day at -15°C and 3 hours at -20°C; and in 15% salt water 3 days at -10°C, 2 days at -15°C and 2 hours at -20°C. Moreover, Kawada (1968) carried out oral administration of Anisakis larvae stored'at low temperature to 18 dogs and investigated .their infection ability (Table V-12). As a result, it was found that the larvae detected in the gastro-intestinal wall and abdominal cavity were all ones which had been kept at 2°C and also that the larvae which had survived in city water for 50 days still had the ability to infect dogs. Therefore, it is judged that refrigeration at about 2°C has no effect on the infection prophylaxis. Gustafson (1953) investigated the effect of low temperatures on Anisakis larvae parasitic on commercial marine fish in Seattle such as herring and salmon. The purpose was to prevent anisakiasis infection of animals. • 212 bred in the zoo, since marine fish are fed to these animals. According to his results (Table V-13), at -5°C and -10°C, most of the larvae died. At -17°C, all died within 24 hours, 93 and at -30°C they were all killed in 5 minutes. As a conclusion, he stated that if herring in 100 lb blocks are frozen at about -30°C for 16 hours, and stored at -12°C, most of the Anisakis . larvae are killed within 24 hours, and even if a few survive they die within a week, so that the fish are safe food hygienically. He reported this for zoo animals, but it can be applied to man. Tanaka (1968) investigated the relation between the freezing temperature and lethal temperature for Anisakis larvae. If an Anisakis larval body is observed through a transmission type biological microscope by setting up a sample cooler, it appears white when not frozen and black when frozen. The freezing temperature of the Anisakis larval body is between -3.5 and -8.5°C, approximately about • -8°C; therefore this temperature will be the lethal tempera- • ture for Anisakis larvae, he stated. However, lethal temperature is complicated, since it is influenced by the activity of the larvae and the time for the temperature to reach -8°C. There are investigation results by Van Thiel (1960) on the resistance of Anisakis larvae obtained from herring to low temperatures. The life span of larvae for combinations 21 3 O of temperatures of -5°C, -3°C, 0°C, 10°C and 20°C and salt water concentrations of 6, 9, 12 and 15 weight per cent, was 21 days in 12% salt water at -5°C, 0°C and 10°C; 28 days in 6% salt water at -3°C; 21 days in 9% salt water at -5°C and in 15% salt water it was 7, 5 and 3 days at 0°C, 10°C and 20°C respectively. Vik (1964) investigated the possibility of anisakiasis. infection as a function of raw fish preparation methods in Norway, and reported that if the fish is frozen rapidly 24 hours before preparation, the infection can be avoided.

-- O El ltiel,. 3 6 m n

1 15 /15 10/10 10/10 10/10 10/10 2 10/10 10/10 10/10 10/10 3 10/10 10/10 10/10 10/10 4 9/10 8/10 7/10 3/10 5 15/15 10/10 10/10 6/10 0/10 6 10/10 8/10 2/10 0/10 1. Number of days.. 7 13/15 10/10 7/10 0110 0/10 8 10/10 7/10 0/10 0/10 2. City water.

9 10/10 8/10 0/10 0/10 , 3 . Salt concentrations (%). 10 14/15 10/10 7/10 0/10 0/10. Results are 11 1C/10 8/10 0/10 13,/10 4. shown as 12 10/10 8/10 0/10 0/10 *Number of larvae surviving/ 13 10/10 9/10 0/10 0/10 Number of larvae used. 1 4 10/10 8/10 0/10 0/20 15 13/15 10/10 10/30 0/10 0/20 16 2/10 0/10 Mark 'F' in the Table 17 4/10 0/10 Ifi 4/10 0/10 indicates the point at 19 6/10 0/10 which freezing occurred. 20 12/1 6 18/20 2/10 - 0/10 25 13/15 30 H/15 11/11 2/10 These notes also apply 35 to Tables up to V-11. 40 9/9' 50• 20/21

Q) keetift4v, ;5( / -C F 11 -(1114e •

Table V-8. Survival time of Anisakis larvae in various concentrations of salt water (2°C). 214

0 e tgo 4 (%) *4* 3 6 10 . 15 ,

0.5 10/10 10/10. 10/10 10/10 10/10 10/10 ( F ) 10/10 10/10 9/10 10/10 2 10/10 10/10 ( F ) 9/10 10/10 9/10 3 6/20 10/10 10/10 9/10 9/10 4 5/10 8/10 8/10 ( F ) 9/10 9/10 5 5/10 10/10 9/10 10/10 .• 10/10 6 1/10 • 9/10 8/10 10/10 10/10

B e 0/10 • 18/30 5/10 10/10 ( F ) 10/10 2 0/10 17/40 8/10 9/10 6/10 3 0/10 15/30 1/10 4/10 6/10 4 0/10 1/30 0/10 0/10 0/10 5 0/10 0/20 0/10 0/10 0/10

Table V-9. Survival time of Anisakis larvae in various concentrations of salt water (-10°C). 1. Hours. 2. City water. 3. Salt concentration (%). 4. Number of days..

e J ( os) Kime *iti* 3 6 10 15

0.5 10/10 10/10 10/10 10/10 10110 10/10 ( F ) 10/10 ( F ) 10/10. ( F ) 10./10 ( F ) 10/10 2 8/10 9/10 8/10 7/10 10/10 3 6/10 8/10 7/10 9/10 10/10 ( F ) 4 1/10 10/10 5/10 5/10 10/10 5 0/10 5/10 6/10 10/10 10./10 6 0/10 9/10 4/10 6/10 10/10 12 0/10 2/10 1:10 1/10 6/10 •Ps 0/10 0/10 0/10 1/10 7/10 2 0/10 0/10 0/10 0/10 2/10 3 0/10 0/10 0/10 0/10 0- . 10

Table V-10. Survival time of Anisakis larvae in various concentrations of salt water (-15°C).

Key as for Table V-9. .215

0. IC) .1 Vo)

3 6 10 15

0.5 12/15 1 11/15 ( F ) 10/10 ( F ) 7/10 ( F ) 6/10 ( F ) 10/10 2 9/15 2/10 3/10 3/10 1/10 ( F ) 3 0 /15 1/10 4/10 4/10 . 0/10 4 0/10 0/10 0/ 5 0/10 0/10 5 0/10 0/10 0/ 5 0/10 0 , 10 6 0/10 0/10 '0/10 0/10 0/10 7 0/10 8 0/10 . 9 0/10 ' (4) a t( 1 0 /10 0../10 . . 0/10 0/10 0./10 2 0/10 0/10 0/10 0/10 0/10 3 0/10 4 0/10 5 0/10 6 0/10 7 0/10 .

Table V-11. Survival time of Anisakis larvae in various concentrations of salt water (-20°C). 1. Hours. 2. City water. 3. Salt concentration (%) 4. Number of days.

216

« eV-12 ":::71.%Drir er 1, 4 24111'.1:ik k ifj

( E ) i , DilJj.titL C ;511-4- )

0 0 CD ° ee ,..t. L. T.: ›.7) M 4.7e 9.) .R .'11. :gek-erzi..05ke (ft 3*4-(3 M) QD ik hI (t ) 111 II 74 ■-. , 1 , %!_2. 'Mg gl w i;R gt 352 15 (e.,c1.1:..r 5 13) 9 2 Ct . C4)- 1 ( D ) 303 10 (71çrt i* (77-1- 13 ) 5 • 304 10 (71plc1.g:_ -(- 10 8 ) 305 10 (2jcitS.71v1, 1.: -r 15 8 ) 3 356 jo .(4* ,p iz. -(- 20 El ) 4 307 10 ( 4rie.1.1:. -C 2513 ) 2 358 10 ( ( ..(121,»4ptz -C• 30 El ) 6 • 3+2 20 1)1\ ;nib r_ -05013 ) eh 1 • 1 313 10 (3%k 111,4,,Pc:r158,7 1 1 ( D ) 1 1 . 314 9 ( 3 "-;,, -fd !1•14'. ,( (.: "c 20 13 ) 1 3:6 Il 317 10 315 10 ( 3 . id IM ,)‘, 1'r:-1- 1 8 ) . 3;0 10 ( 3 ', fi :2"),,1 , 1z. 'r 2 8 ) - 10 318 9 1 3 ',„ fl :2-:.,typg:. -r. 2 8 ) 3IrJ 9 ( 1v,',, 3 0‘4-Pr:. -c2(31 311 - ,., 7 ( 15",,ÉL:k1, .I.g:'r 1 8 ) • • 2 ( D) 35 1 -(-: 10 ( 15',j.:kir, 1-.7. -(113) Table V-12. Number of.Anisakis larvae which had been stored in low temperature detected 24 hours after the infection to dogs. (Mark* in the Table indicates that haemorrhagic spots were observed. D indicates that the larvae were dead.) 1. Dog number. 2. Temperature (°C). 3. Number of larvae administered (survival in days). 4. Number of larvae detected. 5. Stomach wall. 6. Stomach cayity. 7. Small intestine wall. • 8. Small intestine cavity. 9. Abdominal cavity, Small intestine cavity. 10. 15 (5 days in city watel. 10 (7 " tt etc. 11. 10 (15 days in 3% salt watel. 5% 9 9 (20 " It etc.

1 » » 04..S• 1. 0E4 .4084 .0 1 .0'i-4- •0 8 1.8 - 5t 100 77.0 35.0 5.5 -10 86.1 5.0 3.5 0.8 -17 13.9 5.0 0 0 0 -M 0 0

. . (1968) gz..t Table V-13. Percentage of Anisakis larvae surviving at low temperatures (%) 217

1. 30 seconds. 8. 15.0 hours. 2. 1 minute. 9. 20.0 hours. 3. 1 minute 30 seconds. 10. 1 day. 4. 2 minutes. 11. 5 days. 5. 1.30 hours. 12. 10 days. 6. 2.40 hours. 13. From Kagei (1968). 7. 5.0 hours.

(3) Direct Killing Effect of Various Medicines

Kawada (1968), using 28 preparations (12 kinds of helminthic disease remedies, 4 kinds of sulfa drug, 4 kinds of antibiotics, 1 cancer preparation, 4 dyes, 3 others), added 100 units of Trychomycin or Mycostafin, or quantities of the others adjusted to 100Y ( 1 in 10,000 dilution), to 1 le of Ringer's solution, placed larvae in contact with these solutions and made observations at room temperature for 20 days. The results are shown in Figs. V-10 to V-13. Among the 28 kinds of medicine, only . the six kinds Wormin, • Matromycin, Nile blue sulfate, Gentiana violet, Crystal

violet and Methylene blue could completely kill 5 to 10 larvae during the 20-day observation period. Even in Wormin whose effect was the most powerful, it took three days to kill Anisakis larvae completely at 1 in 10,000 dilution. Therefore, we must say that Anisakis larvae are well ' resistant to medicines. 218

e 20134k *Pee Bex in 10 iïetelee eeewp.s4bmeee r,. 2

WOrTilin 10 Erg 3

Sod. santoninic ac id 5 2 Alcopar 5 2 Supatonin 5 5 Emetine hydrochlor. 5 À 4

Dithiazanine iodide 10 ..,iesteet:Peuiregteer= .r.eteeee 10

5 10 15 20 B çt,

Fig. V-10. Direct killing effect of various medicines on Anisakis larvae in vitro. 1. Number of larvae at the beginning. 2. Number of larvae surviving after 20 days. 3. Days.

0 eme) 2013i1UÈ

stitnal 10 .Atffleneemeeteeeeme 7

Fuad in . 5 4 13 ithionol 5 3 Cestcide Bayer 2353 5 4

. Resockin 5' 0011111111111•1111 1 Doming 5 3 Erycon 5 4 Thias in 5 amous. errommormaimm 5 5 10 15 20 a()

Fig. V-11. Direct killing effect of various medicines on Anisakis larvae in vitro.

Key as for Pig. V-10. 219

0 (;) 20sikt, 14,e Acetosulfamin 5 1 à 5

Trichomyc in 5 111111111.1111.■ r 5 Mycostatin 5 2 Tetracycline 5 aimaime mesa 3

Matromycin 5 4 16 0 Nitromin 5 I 1 Nile blue, sulfate 5 mursommaima 10 0 Gentiana violet 5 9 0

5 10 15 20 8

Fig. V-12. Direct killing effect of various medicines on Anisakis larvae in vitro.

Key as for Fig. V-10.

el- 4n o) 20Bk i7( Crystal viole 5 - 14 0 Methylene blue 5 etmunivtansummasem 18 0 Mapharsen 5 amememacommumeurr 3 Sod. arsInobenzol 51 3 Merthiolate 5 1

Control ( Ringer' s sol . ) 10 flr&W.-ge4,:e4leeTaXe_tCue...e•‘4;tilece 8

5 10 15 20 a 0

Fig. V-13. Direct killing effect of various medicines on Anisakis larvae in vitro.

Key as for Fig. V - 10. 220

Methods of Collecting Anisakis Larvae

Although it has no special connection with this chapter, we will in addition describe research on methods of discovery and collection of larval nematodes, mainly Anisakis larvae, from fresh fish. There is a sifting method which is a method of collecting not only nematodes but of small-sized helminths in general, and it is used mainly for collecting helminths from excrement. However, this method cannot be used for Anisakis larvae, since the larvae are parasitic in the musculature and the organs of the abdominal cavity. Koyanagi (1967) examined Anisakis larvae between two glass plates by transmission illumination, after cutting various organs and the musculature of fish into proper sizes. After performing an autopsy of a fish body, Okumura (1967) ascertained the sites of predilection of the larvae by examining the abdominal cavity and various organs macroscop- 2 ically, and then collected the larvae with an anatomical needle taking care not to damage the larval body. For a considerable quantity of fish body musculature, he ascertained the presence or absence of larvae by pressing between glass plates after slicin or by dissecting. Kato et al. (1968) also performed an autopsy like Okumura, then carefully examined 'the pylorus, liver, mesentries, abdominal cavity, 221

etc., and the helminths in the musculature were examined by a 4 to 5 mm glass plate pressing method. Stern et al. (1961) described the rapid counting of Anisakis larvae in salmon by a peptic digestion method. The relative efficiencies of recovery of Anisakis larvae by the dissection method and the peptic digestion procedure with 5 mg of 0.25% pepsin solution per gram of flesh for 15 to 20 minutes at 50°C to 54°C, and also the efficiency of recovery of the worms in the different parts of the fish by the above two methods, were compared. In all cases, peptic digestion proved efficient, rapid, comparatively • simple and suitable for use with large numbers of samples. Oshima et al. (1966) used the peptic digestion method for measuring the parasitic density of encysted Metagonismus ovatus (Yokogawai) larvae which infest ayu-fish (Plecoglossus altivelis Temminck et Schlegel). When fish meat digestion is carried out for the purpose of collecting encysted larvae, 0.01% in 1:10,000 titer pepsin is suitable. If the concentration is higher than this, the amount of precipitate increases rapidly l .and if it is lower t'here is a risk of insufficient digestion. As mentioned above, the macroscopic method, glass plate method, peptic digestion method, fish body self digestion method, etc. are used for investigating nematodes, mainly Anisakis larvae, and trematodes, and needless to say 222

the investigation method varies according to the purpose of collecting the larvae, puch as to collect large or small numbers of larvae, examination in different sites, etc..

Summary

The resistance of Anisakis larvae with due consideration of anisakiasis prophylaxis was summarized above from the.three standpoints of media, temperature and medicines, referring to many research results. Consequently it was found that the resistance of Anisakis larvae to media, heat and medicines is stronger than expected. How much applicable value for the practical point of fish consumption can be obtained from the description summarized above will be • dependent on the results of future experiments based on perfect design of experiment in which the alternative actions of each factor are taken into full consideration. However, in thé practical field, the question of infection prophylaxie is urgent, and the development of countermeasures by making use of salt concentrations, heat, freezing, etc. is desirable at least from the standpoint of cooking or storing methods. We havé .a certain apprehension, namely, whether the pathogenic organ of anisakiasis is always a living • 223 Anisakis larvae or whether it need not be. In other words, is anisakiasis always caused by only live Anisakis larvae ? When larvae are killed by heat or freezing, is there still a danger of the occurrence of anisakiasis if the dead larval bodies or their constituents are left in food ? By consuming these constantly through the mouth, is there any danger of an anisakiasis occurrence being brought on ? If the theory that.aniakiasis is an allergic reaction is considered, it is possible that the dead larvae or the larval constituents could take the role of the allergen. No appropriate diagnosis or therapeutic methods • for anisakiasis have been established. It may be difficult in this context to establish prophylactic countermeasures against this disease. In short, the food hygienic countermeasures against anisakiasis can be established on the accumulation of fundamental research . on larval constituents in connection with the pathogenesis of this disease and of the investigation of larvae killing methods in the practical field.

• • 224 VI. PROBLEMS REMAINING IN ANISAKIS RESEARCH

From the general survey of Anisakis research above, we learnt that although the history of anisakiasis research is still young, it is being pursued actively in the following fields. For instance, the taxonomy and morphology of the larvae and adults of the subfamily Anisakinae and genus Anisakis; ecology and life history of mainly Anisakis larvae and adults which infest and are found in various fish and marine mammals; infection experiments using • experimental animals such as dogs, rabbits, mice and rats; growth and physiology of larvae in vitro and in vivo; , methods of diagnosing this disease by various immunological reactions; investigations of Anisakis disease and helminthic granulomatosis and clinical, pathological research in • connection with such investigations. We learnt that this research is being pursued from wide viewpoints and also that in all of these research areas many questions and problems remain to be solved in the future. In any case, the occurrence of this disease or closely related diseases can be much expected in the future also, and the earliest establishment of diagnostic and • therapeutic methods as well as fundamental prophylactic countermeasures are urgently required. 225 • At present we consider it important that this disease is caused by the consumption of raw fish infested by Anisakis larvae and we wish to pursue the investigation strictly from the food hygienic viewpoint, because it appears that there are only a few research reports in which the prophylaxis or therapeutic countermeasures against this disease were studied from - the standpoint of practical use. We should • like to carry out research on Anisakis larvae from the standpoint of food hygienics, with attention to the following points: - I) Simple methods of taxonomical differentiation of the subfamily Anisakinae larvae p'arasitic on fish. II) Establishment of a method of measuring infection density for larvae parasitic on fish and the investigation of results based on the method.- III) Differences in larvae infection ability due to the freshness and the species of fish parasitized. IV) Differences in chemical composition (mainly proteins, amino acids, nucleic acid substances, enzymes) of larvae and fish components. V) Resistance of larvae (various medicines, food preparation methods, ultraviolet, radiation, electronic stimulation, etc.) 'VI) Infection experiments using live or dead larvae or larval body components (for various fish and • 226 .experimental animals). VII) ImMunological diagnostic methods using fish parasitized by Anisakis larvae (measurement of antibody value of fish towards Anisakis larvae). VIII) Survival or development test of larvae in artificial culture media consisting mainly of extracts from fish, and the conditions. We would like to pursue our research focussing on various questions which are thought to be necessary in the search for anisakiasis prophylaxis from the viewpoint of food hygienics. Of course, we cannot judge objectively 'whether this is the most important question remaining. We have simply expressed our present opinion.

CONCLUSION

We have been able to grasp the present position and progress of Anisakis disease research by referring to the literature below in order to summarize here the food hygienics of Anisakis larvae. Consequently we learnt that this disease is still under research in the medical science field, that 227

appropriate diagnostic and prophylactic methods for this disease have not been put to practical use yet, and that there is almost no research from the standpoint of food hygienics. However, since the consumption of raw fish as sashimi or other dishes has been a general habit of Japanese people for many years, food hygienic research is very important and required urgently as hygienic countermeasures for this habit. In order to pursue research on Anisakis larvae from a food hygienic point of view, we learnt that it is necessary to know the general principles of Anisakis disease, and to grasp the question points which are necessary to devise food hygienic countermeasures against this disease but which have not yet been solved, on the basis of the results of many workers to date. On the basis of the above knOwledge, we wish to commence our research mainly in the practical field of food hygienic countermeasures. Finally we wish to express our cordial thanks to those people' who kindly gave us instruction and assisted in collecting literature for writing this general review. We are also very thankful to Miss Yoshiko Otake who gave us great assistance in writing the final manuscript. 228

REFERENCES

• •• Jagerski aid. L.A. ( 1894 ) : Bait rage zur Kenntnis der Nematoden. es.1 Jahrb. Anat., 7, 449-532.

•• Stiles • C. W. & Hassal. A .( 1899) : Internal parasites of the fur seal.

Report of Fur Seal Investigations. 1891-1897, Washington , Part 3 • 99-177.

• Ba):Iis , H. A.( 1920) :On the classification of the Ascaridae . I . The

systematic value of certain characters of the alimentary canal. Parasitol

12. 253-264.

• Yorke. W. & Maplestone. P. A.(1926 ):The nernatc>de parasites of verebrates. •

I 8: II, 536 B, London ; J & A Churchill Ltd.

•• Baylis, H. 'A. ( 1932 ) : A list of parasitic worms in Ceatacea. Discovery

Reports, 7, 393- 418.

•• Lyster. L. L. (1940) : Parasites of some Canadian sea mammals. Caned.

J.' Research,18( 12 ), 39G-409.

,..1a11311:,Tc( 1940) pi] r: L.) 1.;

-c.- 25( 6.), 728- 742; 28( 7 ), 857-$68.

• Yamaguti, S. (1941 ) : Studies on the helminth fauna. of Japan . Part 35.

Mammalian nematodes . II . Jap. J. Zoo, . , 9, 409-439.

Yamaguti, S. ( 1942 ) : Studies on the helminth fauna of Japan . Part 41,

Mammalian nernatodes.111. Published by the author. Japan .

.. Baylis, 11, A. '1944 ) : Capsularia marina and the Ascaridae of marine

hosts. Parasitol., 36, 119-.121.

• • Johnston. T.11. & Mawson, P. M. ( 1945): Parasit lc nematodes. Rep. Brit.

Austr. Und New ZeaMnd A nta rct. Res. Exped. 1929 - 1931. Series B

and Botany 5. 73 - 159.

• • pollftPo R. Ph.' 1948): Nematode a oesophage sigmoide de lestomac dune

Ores ores ( L., 1789 ) q- ( . Cetace odontocete). Liste des Anisalcis des

cetaces et des pinnipedes . Ann. Parasit . Hum. et Comp., 23.305-322.

•• Vanek, J. (1949) :Gastric submucosal granuloma with eosinophilic

infiltration. Am. J. Path., 25, 397-411.

•• Hitchcock. D.J. (1950): Parasitological study on the Eskimos in the Bethel

area of Alaska. J. Parasita., 36( 3 ), 232 -234. • 2 29

• • Buckley, J. ,I. C. (1951 ) : Immature Porrocaecum recovered from the human -

mouth. Trans. Royal Soc. Trop. .4ied. & Ilyg. ,44, 362.

• • Mosgovoy, A. A. ( 1951 ) : Ascaridata of animals (In Russian ). Trud.

Gelminth. Laborat. AlcacL Nauk. SSSR., 4. 263-269.

•• Feyster, P. (1953) : Über die chirurgisch bedeutsamen Neurome des Magen-

Da rms chlauches. Langen .becks Arch. u. Deutsch. Z .Chir.Bd. ,274, 320 -325.

• Gustafson, P. V. (1953 ) : The effect of f reezing on encysted Anisakis

lztrvae. J. Parasitol., 39( 6 ). 585 -588 .

C) • ( 1954 ) : ill z •j-ze) Wit — ;E- (r•.1 , !IF..1111q: tit], f (11f 336 P.,

0 • • zlIk*.e ( 1954 ) 11.1.(41f, cir) 1 15ill t " fi .11: .11', 54, 10, 940.

•• Margolis , L. (1954) : List of the parasites recorded from sea manunals • caught off the west coast of North America . J Fish. Res. Bd. Cand., 11( 3 ), 267-.283.

•• kkig frk ( 1954 ) C rohn li 4j II flt (1'1 e>1":(i bk, 2 , 7 . 384 -405.

(e) • • ?: JJ ( 1955 ) : L t.: 1 fq• Nt; 1 1;1'i f 3 ( 5 ), 301 -305.

• Puljansky, , Y. I. (1955) : Parasites from the Barents Sea (In Russian). Truci, Zool Inst Akad. Nauk. SSS12,, 19, 5170.

•• Scott • D. M.(1955): On the early .evelopment of Porrocaecum decipions J. Parasitol., 41, 321 -322,

•• Beaver, P. C. ( 1956 ) Parasitological reviews, larva migrans. Expel.

Parasitol ., 5! 6), 587-621.

• .tt.:111i it. • 'Wit' iri" • ( 1956 ): il.e.V1 ?.) 1d! G ranulorna

0 with eOsinophilic infiltration. I I 53, • , ,

230 •

'• Hartwich • C. ( 1957) : Zur Systemat ik der Nernat"den-superfamilie

Ascaridoidea, Zoo!. .16. Syst., 55, 2 11.

• • • .1 l:f• 1Yh • !if( 1957) L . 1755, 25- 38.

•• +-AR ( 1959 ) : Anisakis ') )L i111- fd[31-.... 1 - 1 44H1 Anisakis physeteris cr) If '71 (r)r'll-', • fik 32, 851-870.

04• • .( 1959) Anisakis 0•)Ife (e '4' 3 MM. .A• 2 ebi Anisakis catodonis c7))7:(ei(IFX 114 4:-N.. ).t, 32. 871 - 890.

.. Myers, B . J . ( 1960 ) : On the morphology and life history of Phocanetna

decipiens ( N rabbe, 1878 ) Nlyers, 1959. (Nennitodit , An i saki nne). Can. .1.

Zool., 38, 331- 344,

• • Van Thiel , P . II Kuipers F. C. & Itoskain , 11. 1" 1.“' :,A neinat..le

parasitic t t herring, causing acute abdominal e.yndrorn, in man. Trop. Geogr.

Med.. 2, 97 - 113,

• • Be rfund , B. ( 1061 ) Nematodes f rtan sumo Norwegian marine fishes .Sarsia , 15, 1 - 50.

ED• • fié.ff f:14 • "i:0111;IF/S-( 1961 ) : f1: g:114a) 1 011,. 11, 'ne, 23( 7 ), 686-689.

• Stern • J. A. , Chakravarti , D. , Uzaman , J. R. & Hesselhol t , M. N . ( 1961 ):

Rapid counting of nematoda in salmon by peptic digestion. Bureau of Comm .

Fish . , U . S . Fish and Wildlife Service, SSRF 255. INPFC Document,

Na 180, 1 -4 . •

CI • . IA1 féCs if! III I !Me- • 'J II -fe: • -1.11.).i. • IM ( 196 1 ) : .fil-lV.111:.151 ..e4'll.R. 9141, 23( 8 ), 867-872.

Gaetro- •• Kuipers, F. C. ( 19 (12 ) Ens inof iele flegmone. van de Dllf1110 Da rm. T.

Enterologie, s ( :3 ), 320-327, •

•• Van Thiel , P.11. ( 1962 ) : Anisakias is . Parasitol ., 52 ( 3 • .1 1. 16 - 17.

0 • fg -( 1963 ) : PAO:1 1 1 1 e) 'U. !It Jap. Food Sri 2 • 12. 12 16.

•• Myers, B. J. t 19 63 ) : The migra t of Anisuk is- type In rya... in experimental

animals. Carand..1. Zoo!. , 41( 1 1. 147- 148,

•• ligH IL( 1 0t;3 ) : 1- ;V, 1:1;4 1›, •J4r; !t ;MI r 1,.• T . II !WY: :It II .1: rd; 1- 1)111 IA .J,U3tH, 27.

jit ,,i1 itik • el •Jy... • w. • '49 • -.FT, • ;Ili - - v•-v :Jj ( 1963 ) ; fed }.11 Itiq i fui. .25( 10 ), 1037 - 1041.

• • 1---'11-'42. • 411I 11 rib • ./c( ? c7v.Co4 riz VI 110 • a wive: • en TM 13( .1 ). 325-32e.,

231

• • 11;t1 (1..11.11"1 • )1 I'. /11. 16 I 1: J; !- .t- ; . '!)`• '1,111..2:, 13 ( 7 ), 560.

*• AshbY B • S Appleton • P • -I • & Dawann • I. ( 1:164 ) : Eosinophilic granuloma „ of gastrointestinal tract caused by herring parasite Eus tools rot undo. t um, Brit . Med. J., 5391, 11.11 1145,

?)1 • •.v•Id • h ,c n, i C hi h • ni ioo.i Larva gran ,{ c.r, fired (18) Anisods -type le.,11 ?r1.1011 4'): 14( 7 ), 620 - 621 ..

•• Kuipers , F.. C. ( 1964 ) Eosinophilic phlegnionous inflammation of the

alinicentary canal caused by parasite from the herring. Pathologia et Micro-

- biologia, 27, 925 - 930,

[fil • lyif1I1( 1964 ) Larva migrans ro filf 5.e( 10 )11,kiii- 13 •t4 Ill'ilil#4?4'11.11. Q.) 1.11: IrJ1LL,, 13 ( 4 ), 326-327.

•• Merkelbach, J. W. C. (1964 ) : Een visser met haringwormziekte ( Anisakiasis )

van het rectum. Ned. T. Geneesk, 108, 2131 -2132,

le3 • Pi e- -01( 1964 ) b Larva migrans ( r1e.g. ■"4.1"

• 13( 7 ), 506. •

0 • /:1t 11LX: ( 1964 ) : it 11' T k z4( e1; ),I1 4l: ■'›i-7-1-i1.11.'7, op fig '3'; IV, 532-548.

0 • --k(114.11:- igi • P) /it'll' b'f- • 41111134 M1. • 1§).r1:: ( 196.1 ) : t-,1:eg th v)..p.

■ Yd. 131 7 ),

11111 VS ef • PO • i .romm. rd ( 1964 ) : Larvel miitrans 0['7( 12) Anisak is 6') 4::4;11141, ;•1f fl?..1.11 ■11, 13( 7 ) 589.

t•tnit„.% • Wt. 81;i:41P. ( 1964 ) T k te let ( Anisakis - like larva ) • A V1 1JJ1t, 13 ( 7 ) , 559-660.

«) • E3 e4( 1964) Larva migrans o)1i)f ( 17 ) Anisakis- type !/,), 4-00J- L t.

-tt• (7) 11.e2,.4iki -•&. ef.1-:.111.1&•, 14( 7 ), 620.

•• Vik, R . ( 1964 ) : Anisakis larvae in Norwegian food fishes. Proc. ist Internat.

Congress of Parasitot ., I , 568-569.

0000 i ■ ;• • Asami • K Watanuki , T Sakai 11., Imam,. H. and (ni R. Two cases of stomach granul caused onisakiN- Ilk.. la real nematodes

in Japan. Atn ,. Jour. Trop. Med. & Ifyg • 14( 1 I. 119-- 123.

0 • • f1til %J. +0 • )1' e ( 19.65 ) : 't' 'P k z f LI; 1)41411 44!A tk•Jetk. f.P.1.11,Z, 14( 4 ), 360, 232 •

• il-"e€ • Ill ipiE • I 'is( • +11iim • » • Shlj -111S •14Mii',!;.','i • 51f ti f- • it'e; f".a LA:( 1965 ) : /R)/11:111à eci)(11f'31.,' ( v) 1 ) .IL 1 1:: e ›ib L ts:I'll'e 87K11 ())4/..i'7:(0.1e/?;. iiMe.13( 1 ),23 —30.

20) • fie ire:X.;( 1965 ) : .41i..f1-1:1 14 -4- ..1D ;11, 1 ,_11:.',L'., 14( 4 ), 353- 354.

0 • 1.&41L( 1965 ) : Larva rnigrans (7) MI' ( 19 ) T J.; "1"-!LI.IL.112., 14 ( 7 ) , 621 ,

/.14tYliell • .eUfriS- 05 • • 111 Efuee • teelzum. F__nkize, •

.1-115- • 317111 We • Ili 111.1Ei.:( 1965 ) : Anisak is eflo) AIX tt, 14( 3 ), 230-232. •

(3 • • e+,141( 1965) : PALI L. tz , 2 a) QP.P. -1-- t, 7 113-119.

• • pi lie ( 1965 ) eit.%f( • tPeP tft. 439 P,

(D • DI1n11:_ii43 • fit ,vr, Szt • ./40,11101t: • nVerk( 1965) : • fp.liu.b. 14( 4). 326 - 327,

(3 • *.e.fliF:iPi • Milt • •1•t‘. 1...1 41*.k.( 1965) : Cz ts -C. ',1,f lr.I111.!... 14( 6 ). 542 - 555.

-.-À-.11f111.:ii•ei • ,1•1:: 19'.5 : 11:;?; > F12 f h 1 Ii M.P.:P.. '1& ee.t+1, 2187. 2r, - 30.

(2) e . so p..651 1.,trvs rnigrans :Tr Of 1..:( 14 ) An inak is- typo • •L!se.•,, I.:•:•)te. es- . !It II.. 14' 4 ), 341, C) • III : 1 "r4 ill • II "ri A 44. 7 e_'■ Y1 j • K.: 1.0 I 19 , ■ 51 : Larva m ig ran.% ce> 6Jf ( 13 ) (9. I: .1111 Anteakin- type SC) !it •") Os ;W. --) C . t itiL■, 14( 4), 64 — •

®•enutilre(1.965) : T +/- 4,111 14( 7 ), 639-640.

• nenleigt • **1-#4.z • i=t141( 1965) : T +;114, nef 5E( 1 )44Teei-s.e)reletemetk Steme.:•. e:t elm, 14( 7 ), 606-607,

• .9Z.P. 6I _1(1966):1 T -= xecnetzlkmete -f:. 1it,"•a -.3tervre.*

.,.,PiMGRecutete), 311.

• i/Z.W,e1-1.( 1966 ) : — L -051.14i11q)miter4e-ii--e --D 3.. NnkYtee)12;1-7-. effheitt, 15( 4 ), 31,

• 31 5-( 1966 ) : Larva rnigrans fff 3(21) Anisakis eube.er)

-c. 15( 4 ), 351,

2 233)

C) • - • • 4ft ( 1966 ) : ' '.en11-1: fOrCe... I f1141 "[Pie '3Y, ), 313.

CD■ • 41 tire ( 1966 ) )03 •1›.11;)4.: 21 ( 1 ), 89 - 92.

• 41 "1- !,'1' • f• • 4'1 •0III 91 ( 196G) -Li:if:ill 1 1,1.1th Anisakis 411: .L" 6') Mife. .111..1&'., 15( 4 ), 353-354.

C). :til"- ( 1966 ) : " " 1;4'4 — -Fr 5 ii 14 .11,

(D• 11, 1- • 1.: • • ,1•11, 1 1:', ).-; • ,1-111 Jj • fir:, Ill .il i( 196 (i ) 7,

4 IL. ›..1 1 .1 T ' es WI 1:!ri • . 15 ( 1 ),

92 - 93.

II .1;:, ft 'di pi', if0:à b5111) ( 1966): AI 13,1 - .1 - so t iocyunate v)A„;,.„1,( s

1/-42.1.111; W:e) '1.111..11'•, 15( 6 ) , 507-510:

0 • />1;i • • e> -(1966 ) : T 4t. 7• J. 1LAT ea) 2 !It .bb, 15( 6 ), 484-489,

C) • ,1•I'tlifi -k • ,J•i 11 )1 • R111. I1I 'e M. • 4i )1, 11tGlf.: • Ifif III ( 1966 ) ire P.O.f.1. /f( i; ti 4 Y.7 tit I: t. • -c e) An isak • "i4:g1 ■1)' '1-: !it 15( 4 I. 348-3.19. ■ 0 • ,1•1:;111:1;),.. ( q• !1,11 -- I. !; . 2. ;14. !Il 15 4 3t 31. !pine) 0 • • d•gàlEl.t.k. 11.*.%`.. II I 19(.6 (:,;(

cn41)i-11!:-/: fr.) (Of II 1>:.à. 24( 121, 2314 - 2323,

C) • 1.1 • ,J• ): • lit: III 'All • • • ■ilejl: '4- • 4 i .-)11;44P. • air 1066 ) : onf(1.. If( J; L.:, -4 . O - An isak inae ff.,/ e e)nelM7:rg.14-51*.'pt, .1b, 15( 1 ), 95,

c4 • e.A. led e. • El An( 1966 ) : Larva in igrans cr)(11r- '5( 22) Ç-er (z Anisakis teS1/1.11. ••••*!L .Lb. 15( 4 ), 97 - 98, ■ C) • ie ( 1966 ) : r 'T = -9- k 4, ,. z .1; (1- ea (itiirr In-1 I '11 22, ( 4 ), 556-580,

cD • eF ( 1966): =- 4)- k z t:e St.41.2q:(1'10F11'... ft141 1F-1 1:Ê

. filredfil M.11:e,k( ,e7• ), 312-313,

g • viie +I ( 1966 ) : An isakis 5,)j !I'. ,k 0.11briTn.ç./IfeJell:.114.er Inc (7) ' ?6J Il t • -C ( Larva m ig runs c7)111-/V. 5 ). I '41(1.2-. 22( 4 ) 581-595, • C) • 41- 4e( 196G) : An isakis' type worn; u) 01'11: ( 5 ) .t 9 1iis t. Anisak is cr) L.: is lt 15 ( 4 ) . 96.

0 • ;:à . . );:mi ii; ( 1966) -r ( t HI ogt. Viek â1r15.if . !it 1. 5 ( 2 ), 161- 167.

0 • *i i'*( 1966) : R L flZ e • 5. T f ■ti 15 ( 4 1. 32 - 33,

0 - • )j 1l-: • • V.lut, •%.. ■ ,à1i".%`:; v 1 Pf !4:114.( tà.E.F; 1111 , s. 15( I 1. 154--162.

C": s: P.1 66 4 e \' V.!) !It if

; ry .1* x v,t 'I . ..t . 15( 7 1. 546.

• • '1•1 11111A • I'1 1. • 1 14 011f, !11 • ?9 111'211 1- ,t':f • 1+9. 9c,,; :

11;i (114 1 ei Â p; .7 311 9.

0 • er.4113i.01 • elleam. • • tiq )i • Jr..e., .11i • (3 l'.1 •119661 : !A o) wt.. j.:_jy( 1 ) -r e, U 4 H. ,t) !It .7) 114eii f4: fi .t

%, • • ;S-:!.15.11.1 -ilt, 15( 4 ),. 95 - 96,

C;) • fit'. !PP .),1; ( 1966 ) : Hl. is- • -Z* If( t.: , -C4V; f4;',1.1•. !It if) of • en ddi 11.4 rar 'At, 16 ( 6 ), 543-551. CI • .'(i" LIJEIV/ ( 1966 ) : T .. 'Pt e) e(!.47:tej1ilf '1:.11: 41,, 15( 4 1 . 352.

0 • Ç IJïEilfl (1966) : Anisakis • fdf • 1 • 16 ( 502 - p. et:M(111. ;41-« 6 1, .506.

C) • Ei W-(taq ( 1966) : -9- 7IJJFi - iI1.((L ' 17.) e, 41_

+s- 2£- 1 1 .E> t • 7: ( La i-va inigrans Of Its. 3 ) 22, 486-503.

43) • 11111 .4i" LIU 19661 : L L F J.- 1k •

1 15 (4 ). 29 - 30.

• • f ( 1966 ) : ,Ce IA:1:1We L -C:k.t.14•4:111.v) f fetielf ‘:_

. 1 f5;2; (it) àt. 16 ( 4 1 ,, - 30 •

e • ,i; t4ni...?_( 19661: ) f fL I1t ?.) T J Wink

felier.: -.-3 s .Minophagen Med. Reeler , 11 ( 4 1.. 105-114.

• ( 196i3) : A- FI:AtiD. e) fti'tifP «tie - Nr; J.1414 2204. 10- 16.

g )1f ei:115.b.,11- ,-. 16( 4 295..

• • Ziirrtl • eam.ib.t.=.1.- sot11144 -1)3 ( 1967 ) : 4t. 7. 4 e) 941e 8'..) • A 56IulH lzarq.eue ecell, n. 87 P .

235

s..

e - Ti fe.3.111fk • ( 1967 ) : P T k 7, e_ Ci) IULL1:4114.Pllehlti -):*; Y,i. 4:-'1ifi-fi, 36( 5 ). 663 - 679. • • • ;Min% • eje.r11.1:_:f-(1967):7= 4)- k 7.51ple Lk or) )1/414 fe î (.:>- El 57(10), 1649-1655.

• Ishikura, El. & Kikuchi • Y. (1967): Acute regional ( Anisakiasis) at

Iv.-anai district in Hokkaido. Recent Advances in Gastroenterolo.gy,

11, 444-446. • ■••

Ifee • -figikit( i967) : 5"1 7, 13(11), 429-441.

e • ( 1967 : 5(1.7Ibh• ).0..çtk 3 2 •:--) e) !_É -

• , • AZ el -6-.1£Pili."e'41Ée, 16( 2 ), 85102.

• e • Inà( 1967): tz Anisakis J eg iii? 111. vd ,t..dtstb, 16( 6). .427-435. - •

0• ithât • t4.43:I gt ( 1967 ) : I 1 1- <7) Anisakis fkiZIMI -1" 3 ewe.

e.e. 16( 3 ),•156-166. •

•Ce ••W•têril..1- .. • 1--. 111oik4L. f3- :%( 1967 ) m7 -7:- e>afzUet4(É4i4flel:fitIfifIr2. ,e,11) A, 62(11), 731-736.

• e• 4st.1.91/LX: • /It 111 ( 1967 ) : ;(f!1:_!1111:144:11.1-(4e.i:11,y1- .b elf , k a)he ore/tit: . UI U42J S ?îff&';. !4*(Ik)Æ 320.

4-tte13*' • RX1111.1 - fe411.1( 1967 ) : Ani saki 1C e, tn,mates: Inmetinz ;huge* e!-t. !Ile, 16( 4 ), 289.

0 • 4 , 4143 ( 1967 ) kZ o) ni è /fertb_L mee 61( 5 ). 247-252.

• 4 ,1f41ex 1967 -r !Joel rueferi--fm e!Ë 16( 6). 470-493.

e•mini*.• • re.UI 1967 ) ê -C .1.s 3 -r I MkIn.b. UJII L ezl'!•1118t,tee) 1 ff11. em.-.41.z. 16( 4 ), 292.

, (iD• ett4 fq*.(1967) -: Anisakis 16( 7 - 8

465-497. . . (V • -.):.1:03' ;,X: e:p.p( 1967 ) : Vat .k Ô Anisakis 31.1111.e)1sî4t. ' J.i1i. 16(4 ), 289-290. - - .2. • '' ' • *1:4 W1,:( 167) 7t k .451J1b. c') DiJ14.1. Il42 l.

, . . 322. _

• ? )4611i è.‘zilk •'‘ #1.1/itrfil*f • •Jse)Pet "A. • 1.1 ,Ke. eivol.( 1967 ) : Aniaakia- like ' ilir.,;ee ci) Is-tetittie.f.f.g. ot.rEozut. ezt. 16( 4 ). 288. .... • . . . ,

• • 236 • kiiiEi- tglIat it. • 1 .1 %1Z • è./J1k. 1.1! t';:i 4f ( 1967 ) : T-7- 4,- k. re) M4;I:h•/.4(i.; -D -.C. ;•%•tf.P.i.11,,:.1:,, 16( 4 ) , 294,

(î) • kM11 :.ieei • •1 • MI.11:Th'::; 1.41- • ,1-14111A.:i(.. • 1i*,;;.14•91E..( 1967) : 'Ir.11111:14 e

Jac a) Jiff 3.E. 1 1(i -F9.12 fl'•1`. (11S %ek( N ei: ) k c): ,e7: I , 319. . •

çl • efi 0.1#1:_7_- • .Y-ent • '.•tg- rjE(1967) : k. ,..11É(7)5,14/-e qt":41711. El 4: [K. . iisetti, 2271. 37 - 40. . • (3) • III i;£ tit • .1.« Alit • .(&Yik,t5k. • El ( 1967) : Lamia migrans <7) (I» 23 ) • .1/,111i*.neN Ji it 3 »irt204-1111i c;94einti #4*1- Ili z, Men« • • 0') *et. 'le( 4 ), 296. • 9% • King* • tYi I I ( 1967 ) : 4t- te 31) 41 fe fr- J. z);-'1.flue., 18(4 ) , 293.

• se.itIMZ(1967) . : Ste.84:trfz. Pticece).e, 81( 5 ),. 252-258.

(D • eti eatt • eltrieti.t • itfee • et*tnev1967): 4," ...a)labemeleeteieee,urzeutrelf5e. tiRe42iregt3unsener3u-in(ixer.),t ve,e), 321. , • Plifili1V91(1968) etY210) et. ( Anisakis )eik*M.Ietkg011€. .*Z(Mist10 . • 1:::W -5- •.b se- it de xxif.gi.e.e.• ±.3te • 5)-e,..t

not a in L' (>7cenitetAidMin.S), 3 -11.

0.eine(1968): 7 k iff.',1,-Aliab. 17( 2 ). 255 - 257.

• liumot.• eitsei • .g.stgzn .k• _t-ym • ! Metal{ • fir:dli ef. 1968 ) -r -et •• * J( (1) 1 )4:. Lc:IL H )1z5,S4H2Y-I.fq--eit Lb, 20( 2 ); 175-181.

c-D., e±guat • eitull • 450/11515e(1968): 5), me Éni -at k 51) gi er me -4- ' 3f. gfe.4014.. 17( 2 ). 261 - 262.

• to .wûut • Mitlei I!? • se« OL • 7:.014knJt • 111'n(1968): tz it é 49 - 60. • • xtkiz -D‘% -t. Ei*labliFeletetete. 2e( ),

. Tiff g(1(1968) ; rzt 4i- 7.etz..--)t.‘••C. 1 -{-6-)5,141*.

17( 2 ), 2547'255. •

• eitai • Iîtetti. • esidoe3t • ..e.sife • verenee • i&1Ée5(1968): 7t k ' xgrizAet •E>ef,t( 7 )i*Mlittilk**01*e>51t 1 4-k-i:_1 « .60Aioa")

eAfttle. '17( 2 ), 266.

!,(31 •Ét.rat(1968)zsii,› 7».•)-* 13:4-.5,10:1:. 25(7). 1776.

237

• iituK yr. 6 . • C) • Tier/211(1968) -et

C) • 41;,..11: .11.( 1966 /A t_1( (7) tri. iLlt.. 171 2 ), 258-259,

• 1;1:' 11, tri- 196x f/ , .() f e5

MI, 4 11 5 14 ,

Cf), • ;;«,..-: 11%>1-( 1968 ) 7' •t• f 11:0111. 28. 8 • 1)7 -103,

• 1:,1;%.41% ( 1968 ) Matt ,;) T t. 4 . t!.1 112611

• ( 1968 ) 191!1( 1: lis; f ) • it: 41i 1-1c. flf1ti. 11 )115 I •

• • winos, • il);.q; 93 • ■ 7q it; iv,

ft- za •-• . 7. ‘' 7. .11..41 Vie . i2r 'Lot 18( ).

784- 793.

C ■ • Ilf ee 1, t 11ieni t -r pi ct rt- -r •‘- zJj!; ,. elm rt- -C ( 1 ) 'I. 17 4 3 1 182- 1s6.

• 111 ( .1968 ) : Animaki% e." 6if .3t Aninnk in 5:h 'it e) eit :À-.1>À1*•it'F 26i, 4 ), 224 -244,.

(2) • /1setalatf4 • intE13 akti • ,1•)::, Wf- • A:i it, • 4 1> n • '1'I {)-.:0I( ) : Anisakis Met irx to) il 4i:Itt3' t )"41'•1 17( 2 )• 265 .

C) •à ,i,tlintfÉ • frkrEra--.Eh • •ert.iit • % ex • ,J , A.:1-Z Jalg 11168): */- .W.1,4411141 a te rei .. -f2tZe) ttliet • ',Yee It 141, 1 A L. » ersiEutsituet. e;!1..,..tt.2.. 17( 5 ). 407-413 .

• testettle( 1968) : LIA . -7' 4 , 14( 2 ), 74 - 81,

1968) : T -=- k e)W- 5t ( II ) -= ?F- 7: tint: e) -t3

(t Z., •41i1Z -Dt‘ -C. 17( 5 ), 368-375.

(&;) • *I f III( 1968 ) : 7 P.4j z err

I rrY. tff rt -eI. 7 7 4. . "fee 1:.!11 ■tb, 17( 3). 199-207.

• ft: it • titiFkib • -itIjUe • gra fg( 1968) : 4). C1- 4:51A

I . J31)- A- )e 4 75 c)l11 3. el-A:JAIL 17( 2 ). 262-263.

• k. 1:5 ■1' ( 1968 ) : k 7.« 4f . 21, 95 - 101.

• *111"Eig - :4:5›. • #1.i /Mt • 'M*1:1(1968') : ;fir .,•• it, it

Anisaldnae 47) ffnelit ?fie tfrAJ...elke. Terranova Mel 1:: "Du' 'C. 17( 2 ). 267.

238

(L.2..,e • *.717-MA ( 1968 ).: = -7. A- 7,.e. 5- 4 7, 14( 7 ), 361 - 370.

• $ie*t:: ( 1968 ) : = 4-7- A- .7, e.e)stei?-ità..-itriiiz-i, MI- 3 WI-E. 1. .t 410r/. (7) 'A- Pi. 1J1. 17( 3), 213 - 20.

rlite.:7: (1968) 6- k e“,"E f.1; 1•If -T !I. - 53- Ar./3 ti, 3,EMPkt c: IV.11- blre'-u. • e >7 t- -7 F ( *en-- en+ébireAf'-5). 13 - 20.

• lenl';•;-Zr1 • titti::2_ • • et:t RIN( 1968 ) : Anisakis t' it:at. 17( 2 ), 264. •

4•Kb4ï( 1968) : "efliiketeie)1± L • ei:o•)„,kme-j---- 32( 3 ), 454. e • 1.1" ( 1968 ) : -e ,4r.g.014(7) t G L. teleitE51, 32( 3. ), 449-458. ( 1969 ) ------it A- 7, or) rè -I- (7) Nve. eviwe.. 24( 2),

357-365.. «

tkefitiw:, 24 ( 2 ), 389-400.

( 1969 ) 24( 2 ), 373-374,

• IN t-144( 1969): 4t e) ledli- Ney-:, 24( 2 ), 405 - 412. . - •

• /MT ( 1969 ) : •z-- 4)- k :ys iz »ewe , 24( 2 ), 401-404.

Q-"D • *1.tiE ( 1969 ) : ibiez»,fr e,1112. s.0 tote. let 1 ). 1 -11.

Q-39 • ( 1969 ) : Iteameg .duerie(1.è L A- ) ci)4i4J11-ellter- teneie • -C. kiviee, 24( 2 ), 375-377.

re* .ft(.1969): k Értieltg — (z41LTer,o,5)-gt,- megi t‘ -r. vcikee, 24( 2 ), • 375-377.

(De" Dt111k it L fz.piœsee.-c-1,111 ,no> -r -r ,y F /f.lt tz i3 it -e- „k Ott I& 75( etetc7)/A1"-.1t t Ile .7) MI7-,1« • • Ell tiel**1111 L 239

1. Shioda, H., 1940, 'Hi-tokuisei kaichoen, tokuni kyokushosei 100 kaichoen, kyusei kyokushosei choen ni tsuite (Concerning non-specific ileitis, especially localized enteritis and acute localized enteritis)', Rinsho Igaku (Clinical Medicine), Rinsho Igaku Sha: Misakicho Kanda, Chiyoda-ku, Tokyo.

2. Koizumi, A., 1954, 'Kaichudoku no kenkyu - sono 101 kenky.0 (Research on ascarid poisoning - morphological,

p_yLah.sica.oicalnd_p_hemiçal_restar2h )' , Iwanami Shoten Co., Ltd.: Hitotsubashi, Kanda, Chiyoda-ku, Tokyo. 3. Koshiie, Y., 1954, 'I no mansei enshosei.shuyo no ichirei ni tsuite (Concerning a case of chronic inflammatory tumor of the stomach)', Nippon Geka GakIcai Zasshi (Journal of Japanese Surgical Society), Nippon Geka Cakkai (Japanese Surgical Soc..): 2-5, Kanda Surugadai, Chiyoda-ku, Tokyo. 4. Sunabara, Y., 1954, 'Crohn-byo no byori soshikigaku tek! kenkyu (Pathologic and histological research on Crohn's disease)', Geka no Ryoiki (Domain of Surgery), Ishiyaku Shuppan Co., Ltd.: Komagome, Bunkyo-ku, Tokyo. 5. Motojima, K., 1955, '1 ni hassei shita kosankyu nikugeshu no ichirei (An example of eosinophilic granuloma occurring in the stomach)', • Rinsho Shokakibyogaku (Clinical Gastro-Enterology), Igaku Shoin Co., Ltd:

. 6-20, Hongoe - Bunkyo-ku, Tokyo. 240

6. Ishii, M., Shida, R., Nakano, S., 1956, 'Ihekinai kiseichu shin'nyu ni yoru iwayuru Granuloma with eosinophilic infiltration (A 'Granuloma with eosinophilic infiltration' due to a helminthic invasion into the stomach wall)', Nippon ShokakibpGakkai Zasshi (Japanese Journal of Gastro-Enterology), Japanese Gastro-Enterology Soc.: 2-3, Uchisaiwai-cho, Chiyoda-kul Tokyo. 7. Otsuru, M., Ishizuki, P. Hajikano, T., 1957, 'Yojaku kaichu no choheki meinyu ni yoru kyokushosei choen ni tsuite (Concerning localized enteritis caused by the migration of young ascarid larvae into the intestinal wall) , , Nippon Iji Shimpo (Japanese Medical Journal), Nippon Iji Shimpo-sha: 2-11, Ginza Higashi, Chuo-ku, Tokyo. 8. Teramoto, S., 1959, 'Anisakis no.keitai ni kansuru kenkyu. Dai-ippen: Anisakisphyseteris no keitaigaku tek! • kenkyu (Morphological research on Anisakis. Chapter I: Morphological research on Anisakis physeterie, Kumamoto Igakukai Zasshi (The Journal of the Kumamoto • Medical Soc.), Kumamoto Medical Soc.: c/o Kumamoto Daigaku Igakubu, Honjo-machi, Kumamoto-shi, Kumamoto. 9. Teramoto, S., 1959, 'Aniàakis no keitai ni kansuru kenkyu. Dai-nihen: Anisakis catodonis no keitaigaku teki kenkyu (Morphological research on Anisakis. Chapter II: 24 1

Morphological research on Anisakis catodonis)', KumamotoIgakukai Zasshi (The Journal of the Kumamoto Medical Soc.). 10. Sano, Y., Miyagawa, K., 1961, 'Igan to machigaerareta • iheki kosankyu nikugeshu no ichirei (An example of eosinophilic granuloma of the stomach wall which was mistaken . for stomach cancer)', Geka (Surgery), Nankodo: 3-23, Haruki-cho, Bunkyo-ku, Tokyo. 11. Uchiyama, C., Yamaguchi, K., Aikawa, S., Awaji, T., Maeda, T., 1961, 'Ihekinai kosankyu shinjunsei nikugeshu (Granuloma with eosinophilic infiltration in the stomach wall), Geka (Surgery). 12. Hoshina, R., 1963, 'Gyoniku chu no kiseichu (Parasites 'in fish meat), Japan Food Science, Japan Food Hygiene Assoc.: c/o Kangin Yuraku-cho Shiten, 3L1, Marunouchi, Chiioda-ku, Tokyo. 13. Nishimura, T., 1963, 'Jintai chokanmaku sho-noyo kara miidasareta yojakuna ichi senchu ni tsuite (Concerning a young larval nematode found in a small abscess of the human mesentery), Excerpts . of a talk at the 19th Meeting of the Western Japan Section of the Japan Parasitological Society. 14. Tsuchiya, S., Intikai, S., Utsuki, S., Ito, T., Kuroki, J., Matsuura, K., Ogasawara, M., 1963, Uhekinai kosankyusei nikugeshu no nirei (Two examples of 242

eosinophilic granuloma in the stomach wall), Geka (Surgery). 15. - Asami, K., Imano, H., Watanuki, T., Sakai, G., 1964, Unisakisu ? no kansen ni yoru i no nikugeshu shorei (Cases of stomach granuloma caused by Anisakis ? infection)', Kiseichugaku Zasshi (Japanese Journal of Parasitology), Nihon Kiseichu Gakkai (The Japanese. Society of Parasitology): c/o Kokuritsu Yobo Eisei Kenkyusho, Kami Osaki Chojamaru, Shinagawa-ku, Tokyo. 16. Asami, K., Horimi, T., Inoshita, Y., 1964, 'mu ni okeru Anisakisu shichu kansen jikken (Experiments on infection of dogs with young Anisakis larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 17. Hirao, Y., Yamaguchi, T., 1964, 'Larva migrans no kenkyu (18) Anisakis-type yochu no kakushu dobutsu ni taisuru kansen jikken (Studies of larva migrans (18): Experiments on infection of various animals - with Anisakis-type larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 18. Kunishige, A., 1964, 'Larva migrans no kenkyu (10) Saikin 13-nenkan no byoteki zairyochu ni okeru kiseichusei nikugeshu no kento (Studies of larva migrans (10): Investigation of helminthic granulomas in diseased specimens from the last 13 years) 1 , Kiseichugaku Zasshi (Japanese Journal of Parasitology).

243

19. Okamura, I., 1964, 'Iwayuru Larva migrans (i-senchubyo 104 o fukumu) ni tsuite (Concerning 'larva migrans' (including helminthic diseases of the stomach) ) 1 (Japanese Journal of Parasitology). Kiseichugaku Zasshi 20. Oshima, T., 1964, 'Shokakan anisakAusho (kasho) (Alimentary canal anisakiasis (temporary name) ), Nihon ni okeru Kiseichugaku no Kenkyu (Parasitological Research in Japan). 21. Otsuru, M., Hajikano, T., Hotta, T., Kenmotsu, M., 1964, 'Yojaku kaichuyo senchu no shutoshite shokakan meinyu ni tsuite (Concerning the migration of young larval ascarid-like nematodes mainly into the alimentary canal)', Kiseichugaku Zasshi (Japaneae Journal of Parasitology). 22. Yamaguchi, T., Yanagikawa, H., Kunishige, A., Usuya, N., 1964, 'Larva migrans no kenkyu.(12) Anisakis no kansen shorei (Studies of larva migrans (12): • Cases of Anisakis infection), Kiseichugaku Zasshi (Japanese Journal of -Parasitology). 23. Yoshimura, H., Yokokawa, M., 1964, 'Anisakisuyo yochu • (Anisakis-like larva) ni yoru jinihekinai kosankyusei nikugeshu no shorei (Cases of eosinophilic granuloma in the human stomach wall caused by Anisakis- , like larvae) 1 , Eiseichugaku Zasshi (Japanese Journal of Parasitology). 244

24. Usuya, N., 1964, 'Larva migrans no kenkyu (17) Anisakis-type yochu o toyo shita usagi no soshiki byohen (Studies. of larva migrans (17): Histological lesions in rabbits infected by Anisakis-type larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 25. Asami, K., Horimi, T., Inoshita, Y., 1965, 'Anisakisu shichu no dobutsu kansen jikken (Experiments on • infection of animals with Anisakis larvae) , , Kiseichugaku Zasshi (Japanese Journal of Parasitology). 26. Ishikura, H., Tanaka, S., Goto, T., Aizawa, K., Kanemoto, T., Hagiwara, I., Minamiike, K., Tsuji, Y., Takahashi, N:, 1965, 'Kyokushosei choen no kenkyu (sono ichi) Hokkaido Iwanai chiho ni tahatsu shita jiken 87-rei no ekigakuteki kansatsu (Epidemiological observations in 87 'cases treated by the authors out of many occurring in the'iwanai district in Hokkaido)', Geka Chiryo (Surgical Therapy), Nagai Shoten: 3-111, Kita, Kami Fukushima, Fukushima-ku, Osaka-shi, Osaka. 27. Isobe, M.,'1965, 'Anisakisu aka ni kansuru kenkyu (Studies of the subfamily Anisakinae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 28. Matsuoka, Y., 1965, 'Larva migrans no kenkyu (19) • Jikkenteki anisakisusho ni okeru ketsueki shoken

. (Studies. of larva migrans (19): State of blood in 245

.• experimental anisakiasis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 29. Morishita, T., Kobayashi, M., Sakata,•R., Goto, M., Yamada, I., Sakakibara, H., Mishima, S., Furuhashi, S., Hiraoka, Y., Yamada, S., 1965, 'AnisSkis-sho no hifu han'no (Cutireaction'of anisakiasis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 30. Nishimura, T., 1965, 'Saikin jintai yori miidashita ichi ni no kiseichu yochu no imi suru mono (Implications of some larval helminths found in humans recently)', Mejikaru Karuchua (Medical Culture), Medical Culture: 4-4, Motomachi, Nihonbashi, Chuo-ku, Tokyo. 31. Nishiwaki, S., 1965, Kujirarui, Hireashirui (Cetaceans

and Pinnipeds), Tokyo Daigaku Shuppan-kai (Tokyo University Publishing Corp.):. c/o Tokyo University, Tokyo. 32. Otsuru, M., Hajikano, T., Hotta, T., Kenmotsu, M., 1965, 'Yojaku senchu no shokakanheki meinyusho ni tsuite (Concerning the larva migrans of young larval nematodes in the alimentary canal wall)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 33. Otsuru, M., Hajikano, T., Koyanagi, T., Kenmotsu, M., 1965, 'Yosenchu no shokaki ikosho.ni tsuite(Concerning the larva migrans of larval nematodes in the 246 O digestive tract)', Kiseichugaku Zasshi (japanese Journal of Parasitology), 34. Otsuru, M., Koyanagi, T., 1965, 'Shokaki no iwayuru kosankyusei nikugeshu ('Eosinophilic granuloma in the digestive tract)', Nippon - Iji - Shimpo (Japanese Medical Journal), Nippon Iji Shimpo-sha: 2-11, Ginza Higashi, Chuo-ku, Tokyo. 35. Usuya, N., Shinno, K., 1965, 'Larva migrans no kenkyu (14) Anisakis-type yochu ni yoru kansen jikken (Studies of larva migrans (14): Experiments on infection with Anisakis-type larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 36. Yamaguchi, T., Usuya, N., Hirao, Y., Matsuoka, Y., , 1965, 'Larva migrans no kenkyu (13) Kaisangyo ni okeru Anisakis-type yochu no kisei jokyo to teikosei ni tsuite.(StUdies of larva migrans (13): Parasitic conditions and resistance of Anisakis-type larvae _ in marine fish)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 37. Yasuma, M., 1965, 'Anisakisu-gata senchu no seibutsu- gakuteki kenkyu (Biological studies of Anisakis- type nematodes)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 38. Yokokawa, M., Yoshimura, H., Tsuji, M., 1965, 'Anisakisu-yo yochu kansensho no jikkenteki kenkyu (1) • 247 . • Shodobutsu e no do-yochu kansen to men'eki han'no (Experimental research on infection with Anisakis- like larvae (1): Infection of small animals with the larvae,and their immune reaction)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 39. Asami, K., 1966, 'Anisakisusho no kansen hassho kijo (Causes of the occurrence of anisakiasis infection)', • Showa 41-nendo Mombusho Kenkyu Hokoku Shuroku (Igaku oyobi Yakugaku) (Showa 41st (1966) Research. Report Compilation (Medicine and Pharmacology) of the Ministry of Education). 40. Asami, K., 1966, 'Kiseichusei nikugeshu shutoshite yosenchu no shokaki ikosho ni tsuite. 3. Kansen hassho no inshi (Helminthic granulomas - mainly concerning the larva migrans. of larval nematodes in the digestive tract. 3. Infection occurrence factors)', Kiseichugaku Zasshi (japanese Journal - of Parasitology). 41. Fukunaga, M., Hirao, Y., 1966, 'Larva migrans no kenkyu (21) .Anisakisu-yo yochu no teion ni taisuru teikosel ni-tsuite (Studies of larva migrans (21): Concerning the resistance of Anisakis-like larvae • to low temperatures)', Kiseichugaku Zasshi • , Japanese Journal of Parasitology). 248

42. Inatomi, S., Tongu, Y., 1966, , Kiseichusei nikugeshu no kenkyu (Studies of helminthic granulomas)', Showa 41-nendo Mombusho Kenkyu Hokoku Shuroku (Igaku oyobi . Yakugaku) (1966 Research Report Compilation (Medicine and Pharmacology) of the Ministry of Education. 43. Ishikura, H., 1966, , Kyusei kyokushosei choen (Acute localized enteritis)', .R.insho Geka (Clinical Surgery), Igaku Shoin: 5-29-11, Hongo, Bunkyo-ku, Tokyo. 44. Ishikura, H., Kikuchi, Y., Aizawa, K., Takeda, K., 1966, 'Hokkaido Iwanai chiho'no kyusei kyokushosei choen to Anisakis-sho tono kankei (Relation between acute localized enteritis and Anisakis disease in the Iwanai district of Hokkaido)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 45. Ishikura, H., 1966, "Naman igai naraba anshin - Kiseichu Anisakisu no yoba (No need to worry unless it . isnrae - Prophylaxie of Anisakis helminths)', 'Hokkaido Shimbun, 5-gatsu 14-ka (Hokkaido News,

. May 14). Kagei, N., Oshima, T., Komiya, Y., Kobayashi, A., .Koyama, T., Kumada, M., 1966, 'Sujiiruka no i yori • miidashita Anisakisu-zoku senchu ni tsuite (Concerning nematodes of the genus Anisakis found in • 249 the stomach.of Stenella caeruleo-albus)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 47. Kawashima, K., Hamajima, F., 1966, oyobi Phenyl- isothiocyanate no Anisakis-zoku yochu ni taisuru . satsumetsu koka no kenkyu (Studies of the killing effect of allyl- and phenyl-isothiocyanate on genus Anisakis larvae)', Kiseichugaku Zasshi (Japanese • Journal of Parasitology). 48. Kikuchi, S., Hayashi,.M., Sugiyama, K., 1966, 'Anisakisu-yo yochu shokakan ikosho no nishorei (Two cases of larva migrans in the alimentary canal by Anisakis-like larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 49. Kobayashi, A., Koyama, T., Kumada, M., Komiya, Y., Oshima, T., Kagei, N., Ishii, T., Machida, M., 1966, 'Kaisangyo oyobi ika-rui ni tsuite no Anisakinae yosenchu no kansen chosa (Investigation on infection • of marine fish and cuttlefish by Anisakinae larval nematodes)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 50. Kojima, K., 1966, Œiseichusei nikugeshu - shutoshite yosenchu no shokaki ikosho ni tsuite, 2. Byori soshikigaku tek! shoken (Helminthic granulomas - • concerning mainly the larva migrans by larval nematodes in the digestive tract, 2. Pathological 250

and histological state)', Kiseichugaku Zasshi • (Japanese Journal of Parasitology). 51. Kojima, K., Koyanagi, T., Shiraki, K., 1966, 'Anisakisu-sho (shokakan no kiseichu noya) no byori- gakuteki kenkyu (Pathological research on anisakiasis (helminthic abscesses in the alimentary canal) )' Nippon Rinsh6 (The Japanese Journal of Clinical Medicine), Nippon Rinsho-sha: 3-28, Dosho-machi, Higashi-ku,'Osaka-shi, Osaka. 52. Koyama, T., Kobayashi, A., Kumada, M., Komiya, Y., Oshima, T., Kagei, N., Ishii, T., Machida, M., 1966, 'Kaisangyo oyobi ika-rui yori miidasareru Anisakinae yosenchu no keitaigaku-teki kenkyu (Morphological investigations of Anisakinae larval nematodes which • are found in marine fish and cuttlefish)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 53. Matsuoka, Y., Usuya, N., 1966, 'Larva migrans no kenkyu - (22) Kakusini kaisangyo ni kiseisuru Anisakis-yo yochu ni yoru dobutsu kansen jikken (Studies of Larva migrans (22): Experiments on infection of animals with Anisakis-like larvae parasitic on various marine fish)', Kiseichugaku Zasshi (Japanese . Journal of Parasitology). 54. Matsuoka, Y., 1966, 'Jikkenteki anisakisusho ni okeru 107 ketsueki àhoken (State of blood in experimental 251

anisakiasis)', Shikoku Igaku Zasshi (Shikoku Acta Medica), Tokushima Uiversity, School of Medicine: Kuramoto-cho, Tokushima-shi, Tokushima. 55. Morishita, T., 1966, 'Anisakisu-yo senchusho no men'ekigaku-teki kenkyu (Immunological research on the disease due to Anisakis-like nematodes)', Showa 41-hendo Mombusho Kenkyu Hokoku Shuroku (Igaku • oyobi Yakugaku) (1966 Research Report Compilation (Medicine and Pharmacology) of the Ministry of Education). 56. Shinn°, K., 1966, 'Anisakis yochu ni yoru shokakanheki kosankyusei nikugeshu no jikkenteki keisei ni tsuite (Larva migrans no kenkyu 5) (Concerning the experimental formation of eosinophilic granulomas in the alimentary canal wall with Anisakis larvae (Studies of larva migrans 5) )', Shikoku Igakil Zasshi (Shikoku Acta Medica). • • •

57. Nishimura, T., 1966! 'Anisakis-type worm no kenkyu (5), Kakushu gyorui yori eta Anisakis yochu no ratto ni okeru kansen .taidc (Studies of the Anisakis-type worm (5): Infection behaviour of rats with respect to Anisakis larvae obtained from various marine fish)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 58. Oshima, T., Kagei, N., Kibata, M., 1966, 'Ayu ni okeru yokokawa kyuchu hino yochu no kisei mitsudo sokuteiho - 252

kansen shisu no teisho (Methods of measuring the parasitic density of encysted Metagonismus (Yokogawai) larvae in ayu-fish (Plecoglossus altivelis Temminck et Schlegel) - Proposal of infection index)' Kiseichugaku Zasshi (Japanese Jourhal of Parasitology). 59. Oshima,- T., 1966, 'Kiseichusei nikugeshu shutoshite yosenchu no Shokaki ikosho ni tsuite, 5. Anisakisu no seibutsugaku (Helminthic granulomaS - mainly concerning the larva migrans of larval nematodes in the digestive tract, 5. Biology of Anisakis)', (Japanese Journal of Parasitology). Kiseichugaku Zasshi 60. Otsuru, M., Koyanagi, T., 1966, 'Shokaki no kosankyu shinjunsei nikugeshu (iwayuru kosankyusei nikugeshu) ni teUite (Concerning granulomas with eosinophilic infiltration ('eosinophilic granulomas') in the digestive tract)', Sogo Rinsho (General Clinic), Nagai Pub. Co.: 3-111, Kami Fukushima Kita, • Fukushima-ku, Osaka-shi, Osaka. 61. Otsuru, M., Koyanagi, T., Shiraki, K., Kenmotsu, M., 1966, 'Anisakisu-yo yochu no usagi oyobi mu kansen jikken (Eicperiments on infection of rabbits and dogs with Anisakis-like larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 62. Otsuru, M., Koyanagi, T., Shiraki, K., Hotta, T., Hajikano, T., Kenmotsu, M., 1966, 'Kiseichusei • 253

nikugeshu no kenkyu (Studies of helminthic granulomas)', Showa 41-nendo Mombusho Kenkyu Hokoku ‘ Shuroku (Igaku oyobi Yakugaku) (1966 Research Report Compilation (Medicine and Pharmacology) of the Ministry of Education). 63. Sakumoto, T., Ito, Y., Kusaura, T., Oka, K., Ozaki, F., Inatomi, S., 1966, 'SenChu no kozo to sein i (1), Aji yori erareta yonshu no yosenchu no odanzo oyobi kinkozo ni tsuite (Structure and physiology of nematodes (1), Concerning the transverse section appearance and muscle structure of four kinds of larval nematodes obtained from Trachurus japonicus)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 64. Sano, M., 1966, 'Nihon ni oite . gyokairui ni yotte baikai sareru jintai kiseichu no kenkyu to sono - doko (Research on helminths in the human body carried by marine fish in Japan, and its progress)', • Shokuhin Eisei Kenkyu (Food Hygiene Study), Ministry of Health and Welfare, Food Hygiene Sec., Environmental Sanitation Bureau: Tokyo. - 65. Taniguchi, M., 1966, 'Anisakisu no men'ekigaku tek! kenkyu (Immuncilogical studies of Anisakis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 66. Taniguchi, M., 1 9 66, 'Anisakis no kenkyu, 1. Kogensei IMP (Studies of Anisakis, 1. Antigenicity)', 254

Kiseichugaku Zasshi (Japanese Journal of Parasitology). 67. Usuya, N., 1966, 'Anisakisu-sho no jikkenteki kenkyu kaisangyo kara erareta Anisakisu-yo yochu ni kiinsuru soshiki byohen ni tsuite, (Larya migrans no kenkyu 3) 108 (Experimental studies of anisakiasis - histological lesions caused by Anisakis-like larvae obtained from -marine fish (Studies of larva migrans )', Shikoku Igaku Zasshi (Shikoku Acta Medica). 68. Yamaguchi, T„ 1966, 'Kiseichusei nikugeshu shutoshite yosenchu no shokaki ikosho ni tsuite, 1. Rinsho byorigaku teki kaiseki (Helminthic granulomas - mainly concerning larva migrans of larval nematodes in the digestive tract, 1. Clinical and pathological analysis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 69. Yoshimura, H., 1966, 'Kiseichusei nikugeshu shutoshite yosenchu no shokaki ikosho ni tsuite, 1. Rinsho byorigaku teki kaiseki (Helminthic granulomas - mainly concerning larva migrans of larval nematodes in the digestive tract, 1. Clinical and pathological analysis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 70. Yoshimura, H., 1966, 'Jin shokakan no kosankyusei nikugeshu o kiinsuru Anisakisu-yo yochu ikosho ni tsuite (Concerning the larva migrans of Anisakis-like 255

larvae which causes eosinophilic granulomas in the human alimentary canal)', 71. Yoshimura, H., 1966, 'Anisakisu-yo yochu no shokakan ikosho - tokuni sono rinsho byori (Alimentary canal larva migrans by Anisakis-like larae - especially its clinical pathology)', Nippon Iji Shimpo (Japanese Medical Journal). • 72. Inoshita, Y., Asami, K.,967, 'Anisakisu-sho no kansen seiritsu ni kanyo suru inshi no kento (Investigation of factors which participate in. the establishment of anisakiasis infection)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 73: Ishikura, H., Kikuchi, Y., Aizawa, K., Takeda, K., 1967, 'Anisakisu-sho no jikkenteki kenkyu (Experimental studieS of anisakiasis), Dai 56-kai Nippon Byorigakkai Sokai Shoroku (Excerpts of the 56th General Meeting of the Japanese Pathological Society). • 74.• Ishikura, H., Kikuchi, Y., Hayasaka, H., 1967, 'Cho anisakisusho no rinsho byorigaku teki kosatsu ( Clinical, pathological studies of intestinal anisakiasis)', Nippon Geka Hokan (Archives of Japanese Surgery), Editorial Office of Archives of Japanese Surgery: c/o Kyoto University, Faculty of • Medicine, Yoshida Konoe-cho, Sakyo-kù, Kyoto. 256

75. Ishikura, H., Kikuchi, Y., 1967, 'Anisakisu yosenchu no jintai ikosho ni tsuite (Concerning larva migrans of Anisakis larval nematodes in the human body)!, Nippon Ishikai Zasshi (Journal of the Japan Medical Assoc.), . The Japan Medical Assoc.': 2-5, Kanda Surugadai, Chiyoda-ku, Tokyo. 76. Ishizaki, T., 1967, 'Kiseichu arerugi (Heiminthic allergy)', Modan Media (Modern Media), Nihon Eiyo Kagaku K.K.: 1-33-8, Hongo, Bunkyo-ku, Tokyo. 77. Kagei-, N., 1967, 'Gyorui'kara kansensuru futatsu no kiseichu - sono ekigaku to yobo ni.tsuite (Two helminths which infect via fish - concerning the epidemiology and prophylaxis)', Nippon Eisei Kensa Gishikai Zasshi (The Japan Journal of Medical Technology), Japan Assoc. of Medical Technologists: c/o Kitazato Daigaku, 138, Shiba Shirogane Sanko-cho, Minato-ku, Tokyo. 78. Kagei, N., Oshima, T., Kobayashi, A., Kumada, M., Koyama, T., Komiya, Y., Takemura, A., 1967, ''Kakushu kaisan honyudobutsu ni kisei suru Anisakis-zoku senchu-rui no chosa (Investigation of the genus Anisakis nematodes parasilic on various marine mammals)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 2 5 7

79. Kikuchi, S., Hayashi, S., Nakajima, M., 1967, 'Iruka 109 no Anisakis-sho ni kansuru kenkyu.(Research on Anisakis disease of dolphins)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 80. Kikuchi, Y., Ueda, T., Yoshimoto, K.,.Aizawa, K., Ishikura, H., 1967, 'Cho anisakisusho no jikken menleki byorigaku teki kenkyu (Experimental, immunological and pathological studies of intestinal anisakiasis)', Igaku no Ayumi (Strides of Medicine), Ishiyaku Shuppan K.K.: 32, Komagome Kata-machi, Bunkyo-ku, Tokyo. 81. Kobayashi, A., Kumada, M., 1967, 'Kiseichusei nikugeshu- sho ni kansuru kenkyu, tokuni sono kansengen oyobi shindan ni tsuite'(Studies of helminthic granulomatosis, concerning in particular the source of infection and diagnosis).', Showa 42-nendo Mombusho Kenkyu • Hokoku Shuroku (Igaku oyobi Yakugaku) (1967 Research . Report Compilation (Medicine and Pharmacology) of the Ministry of Education). - 82.› Kobayashi, A., Kumada, M., Hashimoto, K., 1967, 'Anisakis yochu shiyoeki narabini do-chutai chushitsu eki kansaku ni yoru sekkekyu gyoshù han'no (Erythrocyte agglutination reaction due to the sensitization by Anisakis larvae rearing liquid or extract)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 258

83. Kobayashi, A., 1967, 'Anisakisu no bunrui to keitai (Taxonomy and Morphology of Anisakis), Igaku no Ayumi (Strides of Medicine). 84. Koyanagi, T., 1967, 'Anisakisu yochu shokakan ikosho ni,kansuru jikkenteki kenkyu (Experimental research on the larva migrans of Anisakis larvae in the alimentary canal)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 85. Kumada, N., Takeuchi, K., Kubota, H., 1967, 'Ikiteiru Anisakisu I-gata yochu o miidashita kyusei fukusho no ichirei (An example of acute abdomen in which living Anisakis type I larvae were found)', Kisei- . chugaku Zasshi (Japanese Journal of Parasitology). 86. Okumura, T., 1967, 'Anisakis-sho no jikkenteki kenkyu (Experimental research on anisakiasis)', Osaka Shiritsu Daigaku Igaku Zasshi (The journal of the . Osaka City Medical Center), Osaka City Medical Center: c/o University of Osaka City, Fac. of Medicine, 2-29, Asahi-cho, Abeno-ku, Osaka. 87. Oshima, T., 8himazu, T., Akabane, K., 1967, 'Danmenzo ni yoru Anisakis yochu no tokucho (Features of Anisakis larvae in transverse section)', Kiseichu- gaku Zasshi (Japanesa Journal of Parasitology). 88. Oshima, T., .1967, 'Aniàakisu-zoku yochu no odanmenzo ni yoru dotei (Identification of genus Anisakis 259

larvae by their transverse section appearanceP, Showa 42-nendo Mombusho Kenkyu Hokoku Shuroku (Igaku oyobi Yakugaku) (1967 Research Report Compilation (Medicine and Pharmacology) of the Ministry of • Education). 89. Otsuru, M.', Hotta, T., Hajikano, T., Koyanagi, T., Shiraki., K., Kenmotsu, M., 1967, 'Anisakis-like larvae no kakushu kaiaangyorui kensa seiseki (Results of examination of various marine fish for Anisakis larvae)', Kiseichugaku Zasshi (Japanese Journal . . of Parasitology). 90. Otsuru, M., Koyanagi, T., Shiraki, K., Hajikano,.T., 1967,, 'Anisakisu-sho no jikkenteki kenkyu - tokuni sonohassho kiten ni tsuite (Experimental studies of anisakiasis - concerning especially the cause of its occurrence)', Kiseichugàku Zasshi (Japanese Journal of ParasitolOgy). 91. Otsuru, M., Shiraki, K., Hajikano, T., Koyanagi, T., Kenmotsu, M., 1967, 'Kiseichusei nikugeshu no kenkyu (Studies of helminthic granulomas)', Showa 42-nendo Mombusho Kenkyu . Hokoku Shuroku (Igaku oyobi Yakugaku) (1967 Research Report Compilation (Medicine and Pharmacology) of the Ministry of Education). 92. Takayama, T., Hayasaka, H., Ishikura, H., 1967, 'Cho anisakisusho no rinsho to lyori (Clinic and 260

pathology of intestinal anisakiasis)', Nippon Iji Shimpo (japanese Medical Journal). 93. Yamaguchi, T., Kudo, N.,'Sato, K., Usubuchi, I., 1967,- 'Larva migrans no kenkyu (23) Hirosaki Daigaku Igakubu ni okeru saikin 20-nenkan ho byoteki zairyochu ni okeru kiseichusei nikugeshu no kento (Investiga- tions of helminthic granulomas in diseased samples from the last 20 years in the Faculty of Medicine of Hirosaki University)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 94. Yoshimura, H., Arak!, K., Sa!, A., Yokokawa, M., 1967, 'Anisakisu-yo yochu ikosho ni - kansuru kenkyu (Studies of larva migrans by Anisakis-like larvae)', Kisei- chugaku Zasshi (Japanesé Journal of Parasitology). 95. Yoshimura, H., 1967, 'Anisakisu-sho no rinsho byori to hassho'kijo (Clinical pathology of anisakiasis - and the cause of its. occurrence)', Igaku no Ayumi (Strides of Medicine). 96. Yokokawa, M., Yoshimura, H., Tsuji, M., Araki, K., 1967, 'Anisakisu-yo yochu kansensho no rinsho byorigaku teki narabini jikkenteki kenkyu (Clinical, pathological and experimental studies of infection by Anisakis-like larvae)', Showa 42-nendo Mombusho Kenkyu Hokoku Shuroku (Igaku oyobi YakugakU) (1967 Research Report Compilation (Medicine and Pharmacology)

- of the Ministry of Education). • 261

97. Abe, M., 1968, 'Gyorui no Anisakisu (Anisakis) zoku senchu kansen chosa. Shuyo g -,:oshu ni kisei suru Anisakisu-zoku kiseichu no sein, seitai, bumpu oyobi shimetsu genkai ondo nikansuru kenkyu (Investigation on infection - of fish by genus Anisakis nematodes. Studies of the phySiology, ecology, distribution and lethal temperature threshhold of genus Anisakis helminths parasitic in common fish)', Panfuretto (Suisancho Chosa Kenkyu-bu) (Pamphlet, Research Investigation Div., Fisheries Agency, Ministry of Agriculture & Forestry). . 98. Aizawa, K., 1968, 'Anisakisusho no shukushu han'no . (Host reactions in anisakiasis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 99. Hayasaka, H., Ishikura, H., Miyagi, H., Ueno, T., . Utsumi, A., Sato, K., 1968 ; 'Anisakisusho no men'ekigakuteki kenkyu (sono 1) sono hinai han'no ni tsuite

. (Immunological studies of aniSakiasis (No. 1): .Concerning the intracutaneous reaction)', Nippon Rinsho Geka Gakkai Zasshi (The Journal of the Japanese Society of Clinical Surgeons), Nippon RinSho Geka Gakkai: 23, Akefune-cho, Shiba Nishikubo, Minato-ku, Tokyo. 100. moue, T., Miyasato, K., Hosokawa, S., 1968, 'Jikken .teki Anisakisu yochu nikugeshu ni kansuru kenkyu 262

(Studies of experimental Anisakis larvae granulomas) Kiseichugaku Zasshi (Japanese Journal of Parasitology). 101, Ishikura, H., Kikuchi, Y., Hayasaka, H., Miyagi, H., Ueno, T., 1968, . 'Hokkaido ni okeru anisakisusho ni tsuite (Concerning anisakiasis in Hokkaido)', Nippon Rinsho Geka Gakkai Zasshi (The Journal of the Japanese Society of Clinical Surgeons). • 102. Ishikura, H., 1968, 'Anisakisusho ni tsuite, 1. Hassei jokyo to sono rinsho (Concerning anisakiasis, 1. The occurrence conditions and the clinic)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 103. Ishikura, H., Hayasaka, H., Miyagi, H., Ueno, T., Utsumi, A., Saeki, S., 1968, 'Anisakisusho ni kansuru kenkyu (7) Toketsu kanso funmatsu chutai no keiko toyo,ni yoru dobutsu no arerugi han'no ni tsuite (Studies of anisakiasis (7), -Concerning the allergic reaction of animals to which powdered lyophilized larvae were orally administered)', Kiseichugaku • . • Zasshi (Japanese Journal of Parasitology). 104. Ishikura, H., 1968, 'Cho no anisakisusho (Intestinal anisakiasis)', Nippon Rinsho (The Japanese Journal of Clinical Medicine), Nippon Rinsho-sha: 3-28, Dosho-machi, Higashiku, Osaka. 105, Ishikura, H., 1968, 'Anisakisusho ni tsuite (Concerning anisakiasis)', Hokkaido Igaku Taikai Koen Yoshi 263

(Summary . of à lecture at.the Hokkaido Medical Science Meeting). 106.. ICagei, N., 1968, 'Anisakisu-zok-U senchu no seikatsu shi (Life history of the genus Anisakis nematodes)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 107. Kagei, N., 1968 'Sashimi, sunomono goyojin - yàkkaina anisakisusho . (Be wary of raw fish and vinegar pickled. fish - troublesome anisakiasis)', Kagaku Shimbun 4-gatsu 5-ka (Science NeW>s, April 5). 108. Kagei, N., 1968, 'Anisakisu no kansen to yobo (Anisakis infection and its prophylaxis)', Kagaku Asahi (Asahi Science News). 109. Kagei, N., 1968, 'Shingan no kiseichubyo anisakisusho (New visage - the parasitic disease of-anisakiasis)',' ' Hokkaido Shimbun 8-gatsu 26-nichi (Hokkaido News, August 26). 110. Kagei,'N.", 1968,. 'Kaichu ni kawari . shingan zoku zoku - (New faces replacing ascarids appear in quick succession)', Kagaku Shimbun 11-gatsu 5-ka (Science , News, November 15). 111. Kato, T., Kainuma, S., Ito, K., Miura, K., 1968, 'Tokyo Chuo Shijo ni mirareru kaisangyokairui no . Anisakisii-aka senchu ni tsuite (Concerning nematodes of the subfamily Anisakinae found in the marine fish of Tokyo Central Market)', Shokuhin Eisei Kenkyu 264

(Food Hygiene Study). 112. Kawai, Y., 1968, 'Buta kaichu oyobi Anisakisu yochu no yuri-shibosan ni tsuite. (1) Yuri-shibosan (Concerning the free fatty acids of pig ascarids and Anisakis larvae. (1) Free fatty acids)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 113. Kawada, S., 1968, 'Anisakis-sho no yobo ni kansuru kenkyu - Anisakis yochu no teikosei ni tsuite (Studies on the prophylaxis of Anisakis disease - concerning the resistance of Anisakis larvae)', Osaka Ika Daigaku Zasshi (Journal of Osaka Medical College), The Medical Society of Osaka Medical College: 2-7, Daigaku-machi, Takatsuki-shi, Osaka. 114. Kobayashi, A., Kumada, M., Kojima, K., Ishizaki, T., Katsuro, T., Koito, K., 1968, 'Anisakis yochu shiyoeki narabini do-chutai chushitsueki kogen ni yoru hinai han'no (Intracutaneous reactions to the antigens of • Anisakis larvae rearing liquid and larval body extract)', Kiseichugaku Zasshi (Japanese Journal of Parasitology), 115. Kobayashi, A., Kumada, M., Ishizaki, T., Katsuro, T., Koito, K., 1968, 'Anisakisu yochutai chushitsueki narabini do-chutai no haisetsu butsu, bunpi butsu kogen ni yoru hinai han'no. 1. Ippanjin ni okeru hinai han'no seiseki (Intracutaneous reactions to 265

the antigens of Anisakis larvae extract, excrements and secreted substances of the larvae. 1. Results of intracu taneous. reactions in members of the public)', Kiseichugaku Zasshi (Japanese'Journal of Parasitology). 116. Matsumura, T., 1968, 'Kaichu arerugi'(Ascarid allergy)', Modan Media (Modern Media). 117. Nagase, K., 1968, 'Anisakisu no kenkyu (II) Anisakisu . yochu no ratto tàinai ni okeru iko ni tsuite (Studies of Anisakis (II): Concerning the migration of Anisakis larvae in the bodies of rats)', Kisei- chugaku Zasshi (Japanese Journal of Parasitology). 118. Okuno, Y., 1968, 'Buta kaichu oyobi Anisakisu no amino- 112 san ni tsuite. 1. Hakuso kuromatogurafi ni yoru dotei (Concerning the amino acids of pig ascarids and Anisakis larvae. 1. Identification by a thin film'chromatOgraphy method)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 119. Orihara, M., Nanba, K., Kitayama, A., Saito, T., 1968, 'Anisakisu-aka senchu ni kansuru kenkyu.. 1. Hokkaido kinkai ni okeru suketodara to surumeika no chosa (Studies of nematodes of the subfamily Anisakinae. 1. Investigations of Theragra chalcogramma and Ommastrephes sloani pacificus in the coastal waters of Hokkaido)', Kiseichugaku Zasshi (Japanese 'Journal of Parasitology).

266

120. Oshima, T., 1968, 'Anisakisu to anisakisusho (Anisakis and anisakiasis)', Nippon Juishikai Zasshi (Journal of the Japan Veterinary Medical Assoc.), The Japan Veterinary Medical Assoc.:1-39, Akasaka Aoyama Minami-cho, Minato 7ku, Tokyo. 121. Otsuru, M., Shiraki, K., Hajikano, T., Kenmotsu, M., 1968, 'Hokkaido kinkai ni mirareru Anisakinae yochu no keitai kansatsà oyobi kansen jikken, tokuni Terranova yochu ni 'tsuite (Morphological observations on and infection experiments with Anisakinae larvae found in the coastal waters of Hokkaido, concerning especially Terranova larvae)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). 122. Otsuru, M., 1968, 'Anisakisusho (Anisakiasis)', Modan Media (Modern Media). 123. Suzuki, T., 1968,, 'Anisakisusho no men'ekigaku teki shindanho ni kansuru kenkyu. 1. Denki eidoho ni yoru kogen no bunseki (Studies of immunological methods of diagnosing anisakiasis. 1. Analysis of antigens by electrophoresis methods)', Kiseichugaku Zasshi (Japanese Journal of Parasitology); 124. Tanaka, T., 1968, 'Reito ni yoru AnisakiSu no shimetsu. Shuyo gyoshu ni kiseisuru Anisakisu-zoku kiseichu • no sein, seitai, bunpu oyobi shimetsu genkai ondo ni'kansuru kenkyu (Death of Anisakis larvae by 267

freezing. Studies of the physiology, ecology, distribution and lethal teMperature threshhold of genus Anisakis helminths parasitic in common fish)', Panfuretto (Suisancho Chosa Kenkyu-bu) (Pamphlet, Research Investigation Div., Fishei-ies Agency, Ministry of Agriculture & Forestry). 125. Yokokawa, M., Yoshimura, H., Tsuji, M., Araki, K., 1968, • 'Anisakis no men'eki denki eido zo ni tsuite (Concerning the immuno-electrophoresis picture of Anisakis)', Kiseichugaku Zasshi (Japanese Journal of Parasitology). • 126. Yoshimura, H., Kojima, M., 1968, '"Kiseichu-byo no hanashi" chu no •ihappyo deta. (Unpublished data in "Talks on Parasitic Diseases")', Shikai Tembo (Dental . Outlook), Ishiyaku Shuppan K.K.: 32, Komagome Kata-machi, Bunkyo-ku, Tokyo; 127. Yoshimura, H., 1968,. 'Kiseichu-byo no hanashi (Talks on parasitic diseases)', Shikai Tembo (Dental Outlook). 128. Ishikura, H., 1969, 'Anisakisu no hassei jokyo to sono rinsho (Conditions of. anisakiasis outbreaks and the clinic), Saishin Igaku (Recent Medicine), Saishin Igaku-sha: 3-12; Dosho-machi, Higashi-ku,

• • Osaka. 129. Kagei, N., 1969, 'Anisakisu 7zoku senchu no seikatsushi 268

(Life history of the genus Anisakis nematodes)', Saishin Igaku (Recent Medicine). • 130. Kobayashi, A., 1969, 'Hinai han'no (Intracutaneous reaction)', Saishin Igaku (Recent Medicine). 131. Nishimura, T., 1969, 'Aniàakisu yochu no seitai (Ecology of Anisakis larvae)', Saishin Igaku, (Recent Medicine). 132. Oshima, T., 1969, 'Anisakisu no daiichi chukan shukushu ni kansuru kenkyu (Studies of the first intermediate host of Anisakis)', Saishin Igaku (Recent Medicine). 133. Otsuru, M., 1969, 'Kaisan gyokairui kara no - kiseichu- sho. Shutoshite anisakisusho ni tsuite (Parasitic •iseases from marine fish. Mainly concerning

. .anisakiasis)', Shokuhin Eiseigaku Zasshi (Journal of the Food Hygienic Society of Japan), The Food HYgienic Society of Japan: c/o Kokuritsu Eisei Shikenjo, 2-203, Tamagawa Yoga-machi, Setagaya-ku, Tokyo.

134. Shiraki, K., 1969, 'Shokakan yosenchu ikosho (shutoshite anisakisusho) no byori soshikigaku teki shindan ni tsuite (Concerning the pathological and histological diagnosis of larva migrans (mainly anisakiasis) • in the alimentary canal by larval nematodes)', Saishin Igaku (Recent Medicine). 269

135. Suzuki, T., 1969, lAnisakisu no men'ekigaku teki shindan - tokuni kogen no bunri, seisei ni tsuite (Immunological diagnosis of anisakiasis - concerning in particular the isolation and refining of the antigens)', Saishin Igaku (RecentSedicine). 136. References are arranged according to year. Within the same year they are arranged.alphabetically. If there are multiple references by one author - in the .saine year, they are arranged in order of publication. References with two asterisks ** are those which we were not able to refer to directly. 270

A. Contributions to the knowledge of the Nematodes (in German).

B. On the surgically significant neuromata of the alimentary canal (in German).

C. On the taxonomy of the Nematode-super-family Ascaridoidea (in German).

D. Eosinophilic phlegmon of the small intestine (in Dutch).

E. A fisherman with herringworm disease (Anisakiasis) of the rectum (in Dutch).