2. Studies on the Multiplication of Chkunya Virus (CHV), and on Physical and Chemical Structure of Its Virion

PAPER IN SCIENTIFIC SESSION

GROUP A: Immunology and Viral Diseases

1. Principles and Applications of Fluorescent Antibody Technique in Tropical Medicine

Akira Kawamura Department of Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

2. Studies on the Multiplication of Chkunya Virus (CHV), and on Physical and Chemical Structure of its Virion

Noboru Higashi,Yasuko Nagatomo, Akira Tamura and Toshio Ametani Virus Institute, Kyoto University, Kyoto, Japan

3. Electronmicroscopic Studies on the Multiplications of Japanese Encephalitis Viurs

Noboru Higashi*, Toshiko Ametani*, Yasuko Nagatomo*, Eiichi Fujiwara* and Takashi Tsuruhara** *Virus Institute, Kyoto Uniuersity, Kyoto, Japan and **NIH of Japan , Tokyo, Japan

4. Electromicroscopic Studies on the Multiplication of Dengue Virus

Noboru Higashi*, Masao Tokuda*, Toshio Ametani*, Mya-Tu, My My Khin** and Toe Myint** *Virus Institute, Kyoto University, Kyoto, Japan and **Viral Section , Burma Medical Institute, Buruma 72

5. A Plaque Assay of Dengue and Other Arboviruses in Monolayer Cultures of BHK-21 Cells

Hideo Aoki Department of Microbiology, School of Medicine Kobe University, Kobe, Japan

A cell line of baby hamster kidney (BHK-21, clone 13) was found suitable for titration and multiplication of many kinds of arboviruses, including all types of dengue and related viruses. For instance, dengue (type 1, Hawaiian, Mochizuki ; type 2, New Guinea B ; type 3, H-87 ; type 4, H-241 ; type 5 ? Th-36 ; type 6 ? Th-Sman), JBE Nakayama, JaGar # 01, Gl) and Chikungunya (African) viruses are capable of producing clear plaques in monolayer cultures of the cells under a methyl cellulose overlay medium. By means of this plaque assay system, titration and neutralization of these arboviruses are possible. The plaque reduction tests can be made for titration of antibodies. Some basic aspects of arbovirus plaque formation have been studied.

6. Epidemiological Studies on the Transmission of Japanese Encephalitis. Results Obtained in 1968

Yoshito Wada Department of Medical Zoology, Nagasaki University School of Medicine, Nagasaki, Japan

Epidemiological studies on the transmission of Japanese encephalitis have been conducted in cooperation with the Department of Virology, Institute for Tropical Medicine, Nagasaki University, in Nagasaki area since 1965. Mosquitoes were collected at regular or irregular intervals from early spring through late autumn in several villages around Nagasaki City, and Japanese encephalitis virus was isolated from those mosquitoes. Sera of slaughtered pigs were examined for HI antibody throughout a year. These data, together with the record of human encephalitis cases, were analysed. In 1968, the appearance of overwintered females of the vector mosquito, Culex tritaeniorhynchus, started on March 28, and that of newly emerged females started on May 14 ; the number of females was very small in March through May and small in June through August ; the first isolation of Japanese encephalitis virus was made on July 23 ; HI antibody possessing rate in slaughtered pigs reached 50 % in mid-August ; the peak appearance of human cases was seen in mid-September ; and the number of human cases was as small as 20. In comparison with the data obtained in the previous years, it can be said that the starting date for the appearance of overwintered females or of newly emerged females has no relation to the time of Japanese encephalitis epidemic in pigs or in men ; the appearance of infected mosquitoes and the rise of HI antibody possessing rate in slaughtered pigs are seen concurrently ; the size of human epidemic has a 73 positive relation to the number of vector mosquitoes in the period of the rise in HI antibody possessing rate of pigs.

7. Virologic-Epidemiological Investigations on Indonesia III. Comparative Surveys of Anti-Arboviral Antibodies in Sesa Collccted in Indonesia and Japan

Susumu Hotta, Hideo Aoki, Tazuko Yasui and Susumu Samoto Department of Microbiology, School of Medicine, Kobe University, Kobe, Japan

Sera were collected from residents of Indonesia, i. e., Lombok Island in 1964, Lampung State, South Sumatera, in 1965, and Madjalengka State, West Djawa, in 1966 ; as well as from those in Japan, in 1967. HI antibodies against dengue types 1, 2, 3, and 4 (D1, D2, D3 and D4), Japanese encephalitis (JE) , yellow fever (YF) and Chikungunya (CHK) viruses were measured using a microtiter technique (Takatsy-Sever). As to the Indonesian samples, it was indicated : (1) Antibodies against D1, D2, D3, D4 and JE were definitely found. Anti-CHK antibodies were also found, though in smaller percentages. (2) Quantitative distributions of the antibodies were different among the areas examined ; for instance, JE antibodies were com- paratively predominant in the 1964 Lombok sera, D1 and D4 in the 1965 Lampung sera, and D2 and D4 in the 1966 Madjalengka sera, respectively. It was presumed that each particular virus was predominantly distributed in the respective area. (3) Anti-YF antibodies were also detected in sera from all the areas surveyed. Because of their low titers, however, the YF-antibodies were presumed to be due to crossing reactions, especially by dengue infections. As to the Japanese samples, it was indicated : (1) Approximately 60% of the sera tested showed positive reactions against JE, or each type of dengue. No CHK-positive sera were noted. (2) Sera from individuals who suffered from dengue during the 1942-44 Japanese epidemics showed positive reactions against dengue antigens, 20 years after what can be consibered to be a single infection. (3) Although there were sera possessing anti-YF atibodies, this was presumably due to crossing reactions by dengue.

8. Incidence of Bovine HI Positive Reactors to Japanese Encephalitis Virus in Indonesia

Morimatsu Watanabe National Institute of Animal Health, Kodaira, Tokyo, Japan

There are two possible hypothesis about the overwinter of the JEV One is 74 that the virus will hibernate in pig, lizard etc in Japan, and the other is that the JEV will over pass the winter time of Japan in southern warm countries. Japanese research workers showed the possibility of overwinter in lizard and still-born baby pig, while research workers in Okinawa and Formosa demonstrated the incidence of the Japanese encephalitis in pigs in the winter time of Japan. This paper shows the incidence of the disease in cattle, goat and sheep in Indonesia in 1967-1968 by means of serological tests.

Methods Hemagglutination inhibition (HI) tests Hemagglutination (HA) antigen was prepared from suckling mouse brains infected with AS-6 strain of JEV by the method of acetone and ether extract of Clerk and Casals (1958). Test serum was treated with cold aceton and adsorbed goose erythrocytes for removing nonspecific inhibitors and isohemagglutinine. Using as diluent borate buffered saline (pH 9.0) with 0.4 % egg alubumin, serial 2-fold dilutions of test serum were mad in plastic tray and distributed in 0.2 ml amounts. To each well was added 0.2 ml of HA antigen containing 16 units. After incubation at 4 C for overnight, each well was received 0.4 ml of 0.33 % goose erythrocytes suspended in virus adjusting diluent (VAD) of pH 6.6. The test was read after further incubation at room temperature for 60 minutes. Neutralization tests Chick embryo fibro blast cells are used, grown in a medium consisting of Earle's balanced salt solution, 87.5 %; lactalbumine hydrolyzate, 0.5 %; calf serum, 10 %; 7.5 % NaHCO3, 2 % and antibiotics in solution. Serial two- or four-fold serum dilutions are mixed with an estimated 200 plaque-forming units of the JEV in Hank's solution containing 5 % antibody-free calf serum. The mixtures are incubated for 2 hours at 37 C and 0.2 % of each one is inoculated into two drained Petri dishes cultures. After an adsorption period of 90 minutes at 37 C, the mono- layers are covered with agar. The first overlay medium contains final concentrations of 1.0 agar, 0.5 % lactalbumine hydrolysate, 0.4 % glucose, 0.1 % yeast extract and 0.225 % sodium bicarbonate in Earle's solution. After 2 days of cultivation, a second overlay medium containing 1.0 % agar and 1: 10.000 neutral red in Earle's solution is added. Two days later the plaque counts of both dishes at each serum dilution are compared with a number of plaques in the control dishes without antiserum. The serum dilution causing 50 % reduction in plaque count are calcu- lated and expreses the neutralizing antibody titer.

Results The results are shown in Table 1. In Timor island, positive HI reactions were found in 16 cattle among 87 examined, 8 goat among 64 examined and 5 sheeps among 42 examined. In Bali island, all cattle examined, which were harvested from so-called Djembrana disease, showed positive reaction. However, nothing can be said about the correlation between Japanese encephalitis and Djembrana disease. At present, at least it ban be siat that there are many positive HI reactors in animals in Indonesia in the winter time of Japan. 75

Table 1. Positive HI reactors in animals in Indonesia (1967-1968)

Bovine sera were examined by neutralization tests to JEV and also showed positive reaction.

Concludion Incidence of Japanese encephalitis were found in Indonesia in cattle, goats and sheeps in winter time of Japan. These data may suggest that the JEV will over pass the winter time of Japan in warm countries and will come to Japan and cause the epidemics in Japan.

9. The Mode of Development of Japanese Encephalitis Virus in the Vector Mospuito as Observed by the Fluorescent Antibody Technique

Rikuo Doi, Akiko Shirasaka and Manabu Sasa Department of Parasitology, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan

A series of investigations have been carried out on the mode of development of Japanese encephalitis virus in mosquito organs at various time intervals from the infection, by application of direct fluorescent antibody technique on frozen sections of the whole mosquito bodies. JEV virus was inoculated to chicks just after they hatched, and a laboratory strain (YRt) of Culex tritaeniorhynchus summorosus was allowed to feed on the chicks from about 72 hours later, at the peak of viremia. Engorged female mosquitos were collected at certain time intervals from the infection, and frozen sections were prepared with a cold microsome after mounted in a frozen media composed of a mixture of 10 parts of egg albumin and one part of Arabic gum. The sections were treated with anti-JEV fluorescent antibody prepared in this Institute, and the distribution of the specific fluorescence in all mosquito organs was investigated by the direct staining method. The presence of the virus as indicated by the specific fluorescence could be clearly differ- enciated from the non-specific fluorescence seen in various mosquito organs, such as trachea, cuticle and egg shell. Through a series of experiments under various conditions, it has been demon- 76 strated that the virus multiplies first in the midgut epithelium cells in about 5 days after the infection, then spreads into fat body cells of the whole body in about 2 to 3 days, and appears in the salivary gland cells several days later, or from about 12 days after the infection at 25•Ž. Thespecific fluorescence was detected also in other organs, such as in nerve cells, compound eyes, and the Malpigian tubules. A part of the results were published in Jap. J. Experimental Medicine, vol. 37, pp. 227-238 (1967) .

10. Susceptibility of the Lizards to Japanese Encephalitis Virus

Rikuo Doi*, Manabu Sasa* and Akira Oya** * Department of Parasitology, the Institute ofMedical Science, the University of Tokyo, **Deqartment of ViroJogy and Rickettsiology, National Instituteof Health, Tokyo

A series of experiments have been carried out on the infectivity of some Japa- nese lizards to Japanese encephalitis virus, and the role played by them as possible winter reservoirs in the transmission cycle in nature. Two species of the common Japanese lizards, Takydromus tachydromoides and Eumeces latiscutatus, were found to be susceptible to infection of the virus artificially inoculated, causing high titers of viremia for many days, in concentration with their poor antibody producing ability. The lizards kept in cold temperature afterthe infection lost positive viremia, but became positive again after they were exposed to higher temperature levels stimulating to the summer of Japan. Under the labratory condition, the mosquito Culex pipiens pallens was found to feed onthe lizards readily, but the principal mosquito vector Culex tritaeniorhynchus has never fed on them so long as our experiments concern. Since the lizards are efficient predators of the latter mosquito species in their daytime resting places, such as in sweet potato fields, a possibility might exist that the lizards be infected oraly with the virus. The authors consider that the problems of the involvement of some reptiles in the natural transmission cycle of JEV should be investigated more extensively.

GROUP B: Protozoan Diseases

1. Vivax Malaria with Falciparum-Like Temperature Curve

Isao EbisaWa* and Genzo Mitsui** *Institute ofMedical Science, University of Tokyo,Tokyo, Jaqan and **Japan Association Of Troqical Medicine TOkyo, Japan

We are inclined to regard malaria as a disease withanemia, splenomegaly and relatively regular type of fever, par ticuiarly in vivax and quartan malarias. Few young doctors who were graduated from medical schools after the W. W. II have 77

seen malaria in Japan. However, in view of the increasing number of travellers who return from malarious countries by airplaines, we must be prepared for seeing patients of malaria in its very early stage of illness when the signs and symptoms are not typical. We report here a women who recently returned from atrip to Indonesia and New Guinea and stopped taking antimalarial prophylactics immediately when she left endemic areas. Her illness started with two episodes of high fever, one day each per 2 weeks, followed by a daily remittent fever exceeding 40 to 41 C for 5 or 6 days, spontaneous defervescence, and recurrence of the same type of fever. Malaria was suspected weeks after the oncet of fever by her attending physician. She was successfully treated with chloroquine phosphate and primaquine. As regards the treatment, it must be emphasized that primaquine is not registered in the Japanese pharmacopeia and that itcannot be obtained readily, nor can we use it officially when it is obtained.

2. On recent Outbreak of Malaria in Ceylon

Suehisa Takada and Hideo Tanaka Department of Medical Zoology, Osaka City University, Medical School, Osaka Japan

During the period up to 1963 when residual DDT spraying had been carried out, the malaria incidence showed a further decreased in Ceylon and in 1963 only 17 cases were detected. But after the interruption of spray operation in April 1964, gradual increasing of cases occurred from 1964 to 1967. In the latter part of 1967, unexpected malaria epidemics were occurred in Central and North-Central Dry zone, and in early part of 1968 the epidemic assumed explosive proportions with 245,883 positive cases and about 1,000,000 clinical suspected cases being reported from whole island up to30th July of this year. We had an opportunity to make our personal inspection of the epidemic for about three weeks in August of this year by supportof OTCA of Japan and Anti- malaria Campaign of Ceylon (AMC). The present epidemic had its origin in Jungle area in the Central provience, and at first some endemic infections of Plasmodium vivax were mainly detected in new development projects and chena settlements in 1967. At that time, in the endemic district, new gemming area had been discovered in Elahera which attracted people from all part of the country. The infection spread to this area and was carried to all districts by the infected gemmers. With the onset of the North-East Monsoon rains in October, the thousands of abandoned gem pits were converted into a massive favourable breeding ground for the vector mosquito, and by the end of 1967 the malaria had assumed epidemic proportions in Dry Zone. In 1968, the spread of the disease continued extensively and it was further promoted by the mass movement of Buddhist Pilgrims between epidemic area and all parts of island. 78

Thus within a few months the infection become islandwide and now the population at risk to malaria would nearly 9 million, about 75 % of all population, in this country. Parasite formula has changed this year with P. vivax being predominant species followed by some P. falciparum and few P. malariae, and drug resistant strains to Chloroquine are not found. The main vector of malaria in this country is Anopheles culicifacies, and all strains are susceptible to DDT without some partialcases. For present outbreak of malaria, AMC of Ceylon madea new plan of operation to combat malaria epidemic and for eradicationof malaria, with suggestion of WHO, and the plan is carring out actively. All datawere kindly offered by AMC of Ceylon.

3. Studies of Antigenic Heterogenity on Toxoplasma Gondii

Takashi Takayanagi, Hiroshi Kambara and Shozo Inoki Department of Protozoology, the Research Institute for Microbial Diseases, Osaka, Japan

The report dealt with antigenic heterogeneity of four strains of Toxoplasma gondii in the aid of double diffusion method in tubes and of enzymatic degradation by proteases. Whole saline extract of RH strain wasshown 7 precipitates in agar gel layer set with homologous antiserum. They were designated as band a (top in agar gel)•` g (bottom) for convenience. Band b and d were major precipitates. There were 2 common precipitates in all strains used. It was found that the one was corresponded to band b and the other to band d in the precipitate pattern of RH strain. Exoantigen solution derived from RH strain was shown 2 precipitates (band b and d)against anti-RH serum. In enzymatic degradation, proteases (pepsin and trypsin)digested 6 precipitates,but had no effect on a minor antigen (band f).

4. The Effect of Predonin and Eendoxan on Cyst-Formation of Toxiplasma (Beverly Strain) in Mice

Hiroji Kambara, Takashi Tokuyanagi and Shozo Inoki Department of Protozoology, the Research Institute for Microbial Diseases, Osaka, Japan

There are reports indicating that the production ofantibody is a prelude to the formation of cysts in tissues of Toxoplasma-infected organisms. We examined, therefore, how the suppression of antibody formation by the treatment with predonine or endoxan affects to the cyst formation in parasite-infected mice. Mice infected with Beverly strain, avirulent strain, of Toxoplasma took the chronic course of infection. A few parasite appeared in peritoneal exudate only 7 to 9 79 days after inoculation, and the titer of antibody against the parasites measured by hemagglutination test increased from 8 day afterinoculation under our experimental conditions. The cysts in the brain of the infected-mice were observed on 8th day and thereafter increased in number. On the other hand, in mice treated with predonine or endoxan before and after inoculation of parasites, the course of infection was acute, many toxoplasma cells appearedin peritoneal exudate, and all mice died by 15th day. Titer of antibody did not increase at all. However, the cyst formation in brain was observed on 13th day unless mice did not die.

5. Development and Distribution of Cysts in the Brains of Mice Infected with Toxoplasma Gondii

Ichiro Kimura and Toshio Nakabayashi Department of Epidemiology, Institute for Tropical Medicine, Nagasaki Uniuersity, Nagasaki, Japan

A reliable method for diagnosis of toxoplasmosis isthe isolation of the parasite from tissues, of infected men or animals. For this purpose, a mouse inoculation test with spinal fluid or biopsy materials was usually used. On the other hand, the infected brains has been of most use for detecting Toxoplasma parasite in post-mortem examination. In this study done with use of the Beverley strain,examinations were made on the minimum infective number of the parasite contained in cysts, and both development and distribution of cysts in each part of the infected mouse brain, that is, olfactory lobe, cerebrum, cerebellum, interbrain and, pons and medulla.

6. Induction of Specific Resistance to Reinfection of Toxoplasma in Mice

Humio Osaka, Yoshikazu Ok a and Yoshihiro Ito Department of Parasitology, School of Medicine, Tokushima University, Tokushima, Japan

Microorganisms invaded the host, in general, do notseek for host cells for themselves as sites of their proliferation but are captured by a certain group of cells, phagocytes, through which the development ofinfection or the acquirement of resistance initiates. Obligatory intracellular parasites like toxoplasma,however, invade not only phagocytes but every one of capable cells of protein synthesis and this is one of the reasons that the establishment and aggrevation of immunization against toxoplasma is not clearly understood. We experienced a number of fatal and chronic cases when 2 to 40 cysts of low-virulent Beverley strain were given to obtain immunity in mice, and the damage 80 sustained was smaller when mice received 10 to 60 trophozoites of the same strain. None of survived mice from infection with either cysts or trophozoites were able to overcome or withstand the reinfection of high-virulent RH strain organisms. These results differ from those which have been shown by other investigators. Therefore, we pretreated mice with glycogen saline or complete adjuvant before Beverley strain trophozoites were inoculated intraperitoneally so as to have sufficient number of phagocytes in ascites and to be readyto catch out antigenic factors. This application is also seemingly to be advantagesus to curtail the invasiveness of the organisms into cells which are not related to the production of immunity in the host. Responses of the above pretreated mice to reinfection will be discussed here.

7. Experimental Studies on Anti-toxoplasmic Agents

S. Takada, M. Iseki and M. Nishibayashi Department of Medical Zoology, Osaka City University, Medical School Osaka, Japan

1. In case of the administration of phrimethamine (25 mg/kg/day, for 7 days, per os) starting 24 hrs. after infection (104 parasites of Beverley strain, intraperi- toneal), all the treated mice showed neither cysts in brain nor positive HA-test titer on the 37th and 68th day after infection. Subinoculation test was also negative. 2. In case of the administration of pyrimethamine (25 mg/kg/day, for 10 days, per os) starting 30 days after infection, the number 'of cysts in brain decreased considerably and the dye-test titers also declined.However, there was no mouse cured perfectly. 3. The intermittent administrations (Pyrimethamine,20 mg/kg/day, once a week, 7 times initiated 24 hrs. after infection) showed more curative effects on chronic toxoplasmosis in mice than the continuous ones (Everyday for a week, initiated 24 hrs. after infection). 4. SDDS, Sulfisomezole, Sulfamethomizine, Sulfamonomethoxine, Sulfadime thoxine, Pyrimethamine and Spiramycin were found active against acute experimental mouse toxoplasmosis (infected with 105 parasites of RH strain, intraperitoneally).

8. Epidemiological Studies on Toxoplasmosis in Japan

Takeshi Maitani, Toshio Suzuki and Masamitsu Otsuru Department of Medical Zoology, Niigata University School of Medicine, Niigata, Japan

Toxoplasmosis has some characteristic features of zoonoses, in that this pro- tozoa is widespread in many animals all over the world. Although the epidemiology of toxoplasmosis remains unsoluble in many respests, 81 it seems probably that the transmission to human may be caused by contact to suffering animals (direct or indirect), and that some animals living in closed envi- ronment of human beings can play the possible source of infection. In relation to this subject, the authors have carried out to isolate Toxoplasma gondii from various animals by Trypsin-disesting technique since 1964, and obtained some interesting results.

GROUP C: Hookworm Diseases

1. Studies on Ancylostoma braziliense and A. ceylanicum

(1) Morphological differences of both species in adult and infective larva

Yukio Yoshida Department Medical Zoology, Kyoto Prefecture University Medicine, Kyoto, Japan

Ancylostoma ceylanicum has been considered a synonym of A. braziliense by many parasitologists, contrary to the opinions of de Faria who described A. braziliense in 1910 and of Laoss who described A. ceylanicum in 1911. However, Biocca, in 1951, presented evidences that A. braziliense and A. ceylanicum are morphologically distinguishable in the adult stage. After examining this problem from several directions, the present author agreed with Biocca's opinion. In the present paper, morphological differences in adult worms are photographically reviewed, then it is emphasized that both hookworms are also morphologically distinguishable in the infective larval stage.

2. Studies on Ancylostoma braziliense and A. ceylanicum

(2) Results of cross-breeding of both species

Yukio Yoshida, Seiichi Shirasaka, Hiroshi Kurimoto, Sachiko Fukutome and Seigo Okada Department Medical Zoology, Kyoto Prefecture University Medicine, Kyoto, Japan

In order to make sure whether A. braziliense and A. ceylanicum are identical or not, experimental cross-breeding was carried out using A. braziliense of Malay- sian and Brazilian strains, and A. ceylanicum of Taiwan and Fiji strains. The infective larvae of each strain were orally given to pups, then recovered when they grew up to the immature adults. Immature male A. braziliense and female A. ceylanicum, and also opposite combination, were put together in the intestine of another pups. The fact that A. braziliense and A. ceylanicum did not mate, suggests that these hookworms are separate species. 82

3. Studies on Ancylostoma braziliense and A. ceylanicum

(3) Recent survey on their distribution

Yukio Yoshida, Kenji Okamoto and Seiichi Shirasaka Department Medical Zoology, Kyoto Prefecture University Medicine, Kyoto, Japan

Considerable number of reports have been published on spontaneous infection of man and animals with A. braziliense and A. ceylanicum from many parts of the world. However, those reports should be re-examined on their species names because A. ceylanicum had been treated as a synonym of A. braziliense by many parasitologists at that time. After several kinds of investigations the present authors came to the conclusion that A. braziliense and A. ceylanicum are not identical but separate species. Under the knowledge mentioned above, the survey of geographical distribution of these hookworms were carried out in Taiwan, Amami Islands, Fuji Islands, Thailand, Brasil, Malaysia and Ceylon. As a results, A. ceylanicum were found in former four localities, and A. braziliense from Brasil, and both hookworms from later two localities.

4. Studies on Ancylostoma brazilience and A. ceylanicum

(4) Results of oral infection of pups

Yukio Yoshida, Sachiko Fukutome, Seiichi Shirasaka, Hiroshi Kurimoto and Seigo Okada Department Medical Zoology, Kyoto Prefecture University Medicine, Kyoto, Japan

Experimental infection of pups with A. braziliense (Malaysian strain) and A. ceylanicum (Taiwan strain) were carried out in order to compare the biological charactors of both species in the host. In this paper, the results of oral infection are described. Two hundred infective larvae of both species were orally given, using gelatin capsule, to many pups. The animals were killed at various intervals following exposure to infection. At necropsy, all organs and tissues were examined for larvae with the aid of Baermann's apparatus. In both species, the larvae which were swallowed seemed to invade the intestinal mucosa and developed into final 3rd stage. The 4th stage larvae were first recovered on the 2nd day of infection and the 5th stage on the 6th day respectively. They reached sexual maturity about 2 weeks after inoculation. The infection rate was very high in both species as that 70 to 90 percent of given larvae matured. No remarkable differences were found in larval behaviour after oral infection between both species. 83

5. Studies on Ancylostoma braziliense and A. ceylanicum

(5) Results of cutaneous infection of pups

Yukio Yoshida, Hiroshi Kurimoto, Seiichi Shirasaka, Sachiko Fukutome and Seigo Okada Department Medical Zoology, Kyoto Prefecture University Medicine, Kyoto, Japan

Cutaneous inoculations were made by placing 200 infective larvae of A. braziliense and of A. ceylanicum in a few drops of water, for about one hour, on the abdominal skin of pups. The method of examination of animals for larvae was the same as mentioned in report 4. In both species, larvae invaded the skin, migrated to the lungs, then moved to the intestine via trachea, esophagus and stomach. The third ecdysis was seen on 3rd to 4th day of infection and the fourth ecdysis on 7th to 9th day. The worms reached sexual maturity about 2 weeks after inoculation. Although the larval behaviour of A. braziliense in the host was not much different from that of A. ceylanicum, the number of worms matured were much more in the former species than the later.

6. On the Segregation of Ancylostoma ceylanicum and A. caninum in the Host's Intestine

Seigo Okada Department Medical Zoology, Kyoto Prefecture University Medicine, Kyoto, Japan

It is interesting to know the relationship between two species of hookworm synchronously harbouring in a host's intestine. This kind of knowledge must be helpful in considering pathogenicity of A. duodenale and N. americanus when they infect man at the same time. The present author investigated on this problem by means of experimental infection of pups with A. ceylanicum and A. caninum. As a results, it was noticed that these hookworms were inhabiting in the host's intestine under a certain form of segregation. 84

GROUP D: Filariasis

1. Electron Microscopic Studies on Spermatogenesis in the Setaria Digitata

Hachiro Sato, Yoshito Otsuji, Kazuo Yonamine, Mutsuishi Ando, Yoko Sadakata, Tadashi Maeda, Ryuji Harada and Akira Nakashima SecondDepartment of Internal Medicine,Faculty of Medicine, KagoshimaUniversity, Kagoshima, Japan

The purpose of this studies is the observation of the spermatogenesis on the Setaria digitata.

Methods Male worms of Setaria digitata were removed from a peritoneal cavity of the cattles. For electron microscopic observations the worm bodies were fixed for 2 hours in 2 % osmium tetroxide solution buffered with phosphate at pH 7.4 followed by embedding in epoxresin. The material was cut into ultrathin slices on a Por- ter-Blum microtom and doubly stained with uranyl acetate and lead hydroxide to be observed under an electron microscope of JEM-7 type.

Results and Conclusion F. 1 An electron micrograph of the proliferous zone of the testis single layered low lining cells (LC) have many mitochondria, abundant free ribosomes and some dense granules. Round spermatogonin (SG) can be seen in the lumen. BL : Basal lamina. F. 2 Sections of the spermatogonia. The karyoplasm is composed of evenly distributed chromatin granules and a dense round nucleolus. In the cytoplasm the endoplasmic reticulum (ER), free ribosomes and Mitchondria (M) are found, and the Golgi complex are well developed. The mitochondria are abundant as compared with the Dilofilaria immitis. F. 3 An electron micrograph showing a spermatocyte. The spermatocyte is oval in shape, and is about two times larger than the spermatogonium. The nucleus is round in shape. Organelles such as mitochondria, the endoplasmic reticulum and the Golgi complex are more abundand than that of the Dirofilaria immitis. Ovoid bodies (OB) can be seen in the cytoplasm. F. 4 An electron micrograph showing a spermatocyte at a state of metaphase. The nuclear membrane has disappeared and chromosomes (Chs) are already done. In the central portion of the figure is a pair of centrioles (C), and many ovoid bodies (OB) arranged concetrically around the centriloes are found. The bodies are incompletely surrounded by the double membrane structure and are contained moderately dense filamentous substance. F. 5 Spermatids at an early stage of spermiogenesis. In the center of a cell are f ound chromosomes begining to condense. The limiting membrane of the ovoid bodies is more incomplete than that of the spermatocyte. 85

F. 6 Spermatids at a later stage. Chromosomes in cored form are fusing into dense bodies. F. 7 Some nutritive cells and a sperm. The well-developed endoplasmic reticulum and the Golgi complex are found in the nutritive cells. F. 8 A lining cell in the growth zone of the testis. The cell containes dense granules and lipid droplets. F. 9 Mature sperms in testis. The cells are irregular in shape. One or two round condensed chromosomes can be seen in some sperm sections. The mitochondria and the ovoid bodies are pressed against the cell membrane. Contents of the ovoid bodies are reduced. On the contrary, the membrane system of the bodies is remained and convoluted. The cytoplasmic matrix is filled with unevenly distributed amorphous substance.

2. Immunological Time Course in Cotton Rat Filariasis Studied by Complement Fixation Test and Indirect Hemagglutination Test

Akira Ishii, Hiroshi Tanaka, Koichiro Fujita, Masao Kamiya, Hajime Matsuda and Junji Kobayashi Department of Parasitology, the Institute of Medical Sciens, the University of Tokyo, Tokyo, Japan

Basic studies have been carried out on the application of some immunological methods for diagnosis of filariasia, by the use of cotton rats experimentally infected with Litomosoides carinii. The animals were bred free from other helminthic parasites, and certain estimated numbers of infective larvae were inoculated through natural bites of certain numbers of the intermediate host, Ornithonyssus bacoti. Serum samples were collected every two weeks from the infection, and both complement fixation test and indirect hemagglutination test were carried out with the microtiter method. The antigens were prepared from buffered saline extracts of the adult Litomosoides. Sera collected in early stages of the infection were all negative at both tests, while significant increase in CFT titers was detected from 4 to 10 weeks after the infection. The highest CFT titers observed was 128 X. No significant correlation was seen between the CFT titers and the density of microfilariae in blood samples. The increase in HA titers was seen also from 4 to 6 weeks after the infection, and the highest titer recorded was 32,800 X. The heterophile agglutinating antibody against sheep erythrocytes was not detected in the cotton rat sera. 86

3. Studies on Antibody Formation in Cotton Rat Filariasis Sequential Appearance of 19 S and 7S Antibody in Cotton Rats Infected with Cotton Rat Filaria, Litomosoides carinii

Koichiro Fujita, Junji Kobayashi, Hiroshi Tanaka and Manabu Sasa Department of Parasitology, the Institute of Medical Science, the University of Ttokyo, Tokyo, Japan

Through our previous studies, it has been demonstrated that cotton rats infected with Litomosoides carinii produce antibody against the adult worms, that become detectable at least several weeks after the infection. In the present study, serum samples were collected by total bleeding of cotton rats at various time intervals from the infection, and the hemagglutination titers against crude extracts of adult worms as the antigen were examined after fractionated by gel filtration with Cephadex G 200. The sera collected within 6 weeks from the infection were all negative, while the antibody activity became first detectable in 19 S fraction from the seventh week, and remained positive restricted to this fraction during the period to the 11.5th week. The antigenic activity in 7 S fraction became positive from the 12th week, and became completely restricted to this fraction from after 14.5 weeks. These facts were confirmed also by treating the serum samples with 2-mercaptoethanol. It has further been demonstrated that the first peak of the titers in Sephadex G 200 gel filtrates corresponded to 19 S and the second to 7 S by both the ultracentrifuge method and the isotope labelled antibody analysis method.

4. The Mode of Action of Diethylcarbambzine on the Cotton Rat Filaria, Litomosoides carinii

Junji Kobayashi, Hajime Matsuda and Manabu Sasa Department of Parasitology, he Institute of Medical Science, the University of Tokyo, Tokyo, Japan

Diethylcarbamazine (DEC) is a drug efiective against filarial parasites, and has been extensively in the treatment and control of human filariasis. It has remarkable effects in killing microfilariae in circulating blood, both in human and animal filarial infections, and was shown in our previous experiments to be effective also against adult worms of the cotton rat filaria when large doses were administered. However, little has been known on the mode of effects of the drug against the parasites. The drug has no direct filaricidal effects in vitro both against microfilariae and adults in culture media. A series of laboratory experiments were carried out on the mode of effects of DEC on various stages of the cotton rat filaria under various conditions. The periods of survival of microfilariae, infective larvae and adult worms in culture media containing various concentrations of DEC did not differ significantly from 87 those kept in DEC free culture media. On the other hand, DEC injected intraperitoneally at sufficient doses (such as 20 mg/kg or higher) was effective to reduce microfilarial density of the host blood to the minimum levels at least for a number of days, and large numbers of the microfilariae were found to be trapped and killed in liver capillaries at autopsy, Microfilariae collected from in vitro culture media of the female worms and injected into clean cotton rats were found to cause microfilaremia, but DEC injected into such non-immune animals caused no effects in reducing the microfilarial levels. However, it was further demonstrated that DEC injected into such animals with artificially induced microfilaremia was effective in reducing the blood level when immune sera collected from the infected cotton rats were injected prior to the drug treatment, though such effects lasted only for a short period. Microfilaremia in non-immune animals could be produced artificially also by surgical transplantation of the adult parasites into peritoneal cavity of clean animals, but DEC again had no no microfilaricidal effects when injected into these animals 5 days after the transplantation.

5. Results of the Skin Test for Filariasis Carried Out in Hachijokoshima, the Endemic Place of Malayan Filariasis and in Otogane Area, Fukushima which is Free from Human Filarial Infection

Shigeo Hayashi, Hisashi Yamamoto and Ryoko Sawadaichi Department of Parasitology, School of Medicine, Yokohama City University, Yokohama, Japan

The skin test for filariasis has been demonstrated to be useful in the epidemiological survey for the assessment of the status of transmission. So far, however, the test has been applied mainly in the areas of bancroftian filariasis and few trials have made for malayan filariasis. Herewith presented are the results of the tests using Sawada's FST Antigen which were carried out on the inhabitants in Hachijo-Koshima, the only endemic place of malayan filariasis in Japan. It was observed that the majority of the in habitants, that is, 33 persons out of 39 or 84.62 % reacted positively to the tests. On the other hand the results of the same test on the school children in Otogane Area, Fukushima where no human filariasis known existing showed that only 10 out of 195 or 5.13 % of the examinees reacted positively. These might be considered as the false positive reactions. Therefore, the specificity of the test was worked out as 94.87 %. Both in Hachijo-Koshima and Otogane area the fecal examinations for the infections with intestinal helminths were carried out. The infections with Ascaris, Hook worms, Trichostrongylus and Trichuris were fourd, however, there were no statistically significant associations observed between the skin positivity and other helminthic infections. 88

6. Epidemiology of Transmission of Bancroftian Filariasis in Vector Mosquitoes Under Various Levels of Endemicity

Nanzaburo Omori Department Medical Zoology Nagasaki University School of Medicine, Nagasaki, Japan

The object of this experiment is to learn how is the reduction of the natural infection rate of mosquitoes under the gradual decrease in the level of endemicity of bancroftian filariasis in a village, and to learn why the rate is lower in Ae. togoi than in C. p. pallens. Okubo is a farm village located in Uku Island, Nagasaki prefecture. It had 54 houses and 284 population in July, 1963 when the blood examination for filariae was made by prefectural and 66 persons or 25.3 percent of those examined were found positive. The positives were advised to take the drugs, diethylcarbamazine on May of 1964. Four months later, we started to examine the states of endemicity of the disease and natural infection of vector mosquitoes. Every year, mosquito surveys were made 4 or 5 times during from late June to early August and thereafter rdsidual spray for all houses and blood examination by 60 mm3 blood for all persons were made. As a result, it was found that the microfilarial density per positive person per 60 mm3 blood decreased from 87.4 in 1964 to 26.1 in 1968, and the microfilarial prevalence of the village decreased from 26.4 % in 1964 to 12.7 % in 1968. With the decrease in the microfilarial prevalence of the village, the natural infection rate decreased by negative acceleration in the case of C. p. pallens and it decreased much more intensively in Ae. togoi. The reason of being lower in Ae. togoi in natural infection rate seemed to be due to the fact that the pick-up number of microfilariae is generally much smaller in Ae. togoi and this seemed to result in the lower infection rate in this mosquito especially when fed on carriers of lower microfilarial counts.

7. A Case of Pulmonary Dirofilariasis Causing Acute Infarct of the Lung

Hiroyuki Yoshimura *, Muneo Yokogawa *, Ryokichi Momma **, Kazuo Yamato ** and Akio Bukawa ** * Department of Parasitology, School of Medicine, Chiba University, Chiba, Japan and ** Department of Surgery, National Kanazawa Hospital, Kanazawa City, Japan

It is the purpose of the present paper to report the first case of pulmonary dirofilariasis from Japan. Case Report : A 42-year-old, high school teacher, living in kanazawa City, Japan. When he rose in the morning on February 27, 1968, he suddenly fell into a faint for a while. Chest X-ray examination taken on March 6 demonstrated a 89

rounded 2 cm density to be in the lower lobe near by the diaphragm. Preoperative diagnosis was probable tuberculosis or granuloma, although a malignant tumor could not be ruled out. The total leucocyte count was 8,400 with 6 % eosinophils. Intradermal test with Dirofilaria immitis antigen was positive (3o •~ 40 mm in diameter) but negative with Paragonimus westermani antigen. On April 17, 1968, a thoracotomy was performed to resect the involved area. The nodular focus measuring 2.2 •~ 1.4 •~ 1.3 cm showed typical hemorrhagic indura- tion of the lung. Microscpically the transverse or obliquelongutidinal sections of the worm were found in the lumen of media-sized pulmonary arteries showing organization of thrombi. Parasite : The transverse sections of the worm ranged in diameter approximately 0.3 •~ 0.7 mm. The thick three layered cuticule without longitudinal external ridges, broad lateral chords, well developed muscle layers, internal longitudinal cuticular ridges expanding inwardly at the lateral chords were disinguishable. A

genital organ most probably to be ovary was present. The nematode was identified as possively young but sexually matured female worm of Dirofilaria immitis.

8. Filariasis in Brazzaville, Congo

Toshio Yamamoto, Hiroshi :Takahashi, Hiroyuki Amano, Toru Yamanaka and Masanori Aihara Tenri Hospital, Tenri, Miie, Japan

Twenty-one cases of Filariasis, treated in our clinic in Congo, were classified into 1 Filariasis bancrofti, 13 Loiasis, 6 Acanthocheilonemiasis and 1 unclassified one respectively. Clinically, elephantiasis was found in Filaria bancroft case and 2 Fye-warms were discovered in Loiasis. The other specific symptoms were not obtainable. Hematologically eosinophilia (average 20 %) were found in all cases. In addition, slight left on granulocytic series were noted in Acanthocheilonemiasis. The other findings were within normal limits. In Filariasis bancrofti and Loiasis microfilaria in the peripheral blood were disappeared and their symptoms were improved during the course of Diethylcarba- moyl-4-piperazine administration, whereas no improvement was noted in Acantho- cheilonemiasis. 90

9. Lymphangioadenopathy of Filarial Chyluria

Analysis of Lymphaticopelvic Fistulization

Shokichi Tani * and Masayoshi Akisada ** * Department of Internal Medicine, The Institute of Medical Science, University of Tokyo, Japan, and ** Department of Radiology, Faculty of Medicine, University of Tokyo,

Filariasis is a disease of lymphangioadenopathy of the infested site of the body. Clinically there are two different symptoms in the chronic stage, that is chyluria and elephantiasis. Etiological mechanism of chyluria is only pertially understood. Therefore, lymphography is a usefull method to Know pathophysiologic state of filariasis, especially in chyluria. We surveyed 34 patients of filarial chyluria by Kinmonth's method of lymphography to research it's mechanism. Summary of the results as follows. 1) Lymphograms of 32 out of 34 cases (94.1 %) of filarial chyluria specifically showed lymphaticopelvic fistulization. This finding is characteristic of filarial chyluria. 2) The lymphaticopelvic fistulization in the lymphograms was classified into the following six types : a) systic type ; b) circular or semi-circular type ; c) vermiform plexus type ; d) arborization type ; e) indeterminate type ; and f) type without fistulization. The former three types indicated the more advanced stage of filarial chyluria the latter three types. 3) From the relationship between the lymphaticopelvic fistulization and the abnormalities of the para-aortic lymph nodes or of the thoracic duct, the pathogenesis of Ethiodol reflux into the collecting lymphatics of the kidney was suggested. In other words, lymphaticopelvic fistulization may be caused by the abnormalities of the lymph nodes associated with abnormalities of the lymphatics in the para- aortic region on the same side. 4) On the therapeutic effects of lymphography on filarial chyluria, Kinmonth's method may be one means of treating chyluria. But further follow-up study is needed to conclude it's therapeutic effect Here, we suggest that lymphography is clinically available for diagnosis of filarial chyluria by analysing the type of lymphaticopelvic fistulization and the features of lymphangioadenopathy of the lymphatic trunks. 91

GROUP E: Miscellaneous Nematoda Diseases

1. The Incidence of Angiostrongylus cantonensis Among Achatina fulica in Syrian District, Sarawak, Malaysia

Shozo Inoki, Tadamasa Ono, Hiroji Kambara, and Takeo Itakura Japan ResearchInstitute for Microbial Diseases,Osaka University,the 5th Medical Missionof Osaka Universityto Sarawak, 1967

In August 1967 the 5th Medical Mission of Osaka University to Sarawak made epidemiological survey of parasitic diseases in Sirian District, Sarawak, Malaysia. Then we examined the incidence of Angiostrongylus cantonensis among Achatina fulica. In Kuching District the investigation of rodents was made by J. F. Alicata in 1966, but infection rate of its intermediate host has not been inquired yet. By the result of our survey, it was suggested that the infection rate of the larva is high at least in Sirian.

2. Preliminary Observations on the Pathogenesis of Geographic Strains of Angiostrongylus cantonensis in the Taiwan Monkey (Macaca cyclopis)

John H. Cross and James W. Fresh U. S. Naval Medical Research Unit No. 2, 7-1 Kung Yuan Reed, Taipei, Taiwan, Republic of China

Human infection with the metastrongylid lung-worm of rats, Angiostrongylus cantonensis, has been known to occur on Taiwan since 1944 when S. Normura and R. H. Lin found actively moving worms in the spinal fluid of a 15-year-old boy diagnosed as having meningitis. Following Rosen's report in 1962 on the importance of this parasite as a cause of eosinophilic meningeencephalitis in humans, an increasing number of cases of human angiostrongyliasis has been found on Taiwan. Although the number of cases of human disease has not been as high on Taiwan as in other areas, such as Tahiti and Thailand, an extremely interesting aspect of some of the cases has been the recovery of living worms from spinal fluid. Since this finding has not been reported from patients in other endemic areas it was thought that the Taiwan strain of A. cantonensis might possibly be different from strains found in other countries. A study has therefore been initiated to determine whether differences existed between geographic strains of the parasite. The present report concerns preliminary observations of the pathogenesis of six geographic strains of A. cantonensis in the Taiwan monkey, Macaca cyclosis. Strains of A. cantonensis have been obtained from Taiwan. Hawaii, Thailand, Philippines, Indonesia and Malaysia and maintained in the laboratory by passage through the aquatic snail, Australorbis glabratus, and Long-Evans rats. 92

Initial studies were carried out using the Taiwan strain of the parasite. Ten Taiwan monkeys given oral infections of 10,000 larvae died between the 7th and 35th days of infection. All the monkeys lost a considerable amount of body weight, they developed a leucocytosis, and most exhibited an eosinophilia. Spinal fluid was found to be cloudy and contained lymphocytes and eosinophiles. At autopsy the lungs were atelectated and there was vascular congestion of all organs. The central nervous system was also congested. Lymphocytic and mononuclear cell infiltration into the cerebral and spinal leptomeninges and also the pia and arach- noid was evident. Worms were found in the leptomeninges, brain, and cord substance as well as the Central canal of the spinal cord. Pathologically, the parasites appeared to produce focal hemorrhages and abscess in early infections and in older infections, granulomatous space-occupying leisons. Worms were not found in any other organs. While the pathogenesis of the other geographic strains has not been studied as extensively, early results indicate that the Hawaiian, Thailand, Philippine and Indonesian strains of the parasite are equally as pathogenic as the Taiwan strain. Thus far two monkeys each given 10,000 larvae of the Philippine, Thailand, and Indonesian Strains died 6 to 14 days after infection, and four monkeys that received the Hawaiian strain died 6 to 16 days following infection. The pathological findings in these animals were similar to these seen in monkeys receiving the Taiwan strain. The Malaysian strain, on the other hand, has not produced fatal infections. Alth- ough the semen animals tested developed symptoms, and immature worms were recevered in the spinal fluid of one of the monkeys, all animals survived the infections. The results obtained clearly suggest that the Taiwan, Hawaiian, Thailand, Indonesian, and Philippine strains of A. cantonensis are highly pathogenic to the Taiwan monkey while the Malaysian strain is not. It is worthy to note that the pathological findings in the monkeys that succumbed to the infection are similar to those that were found in a child that died of the disease in Taiwan.

GROUP F Trematode Diseases

1. Fine Structure of Schistosoma japonicum

Seiichi Inatomi, Daigoro Sakumoto, Yasumasa Tongu, Setuo Suguri and Kazuo Itano Department of Parasitology, Okayama University Medical School, Okayama, Japan

The purposes of this study are to get the normal pattern of the fine structure of Schistosoma japonicum and of the chaining after treated with anthelmintics. 93

Method The living worms, which were collected from portal veins of experimental hamster about ten weeks later after infection and ten days after treated with anbilhar, were fixed with phosphate buffered 1 % cold gultaraldehyde solution (pH 7.4) for 30 minutes and were washed well with phosphate buffer. After that, they were post fixed for 2 hours in phosphate buffered cold 2 % osmium tetroxid solution and embedded in Epon 812 after dehydration with ethanol series. The spacements after sectioned with Porter-Blum ultrmicrotome were stained with uranyl acetate and lead nitrate. Results The body surface of Schistosoma japonicum is covered with anucleated integument, which has the undulatory spong like structure. On its ventral surface spines sparsely growing, and in some regions there are many spines gathered in group. In male, the dorsal portion has no spine, and in female the number of spines on the whole is less than in male. In the integument are seen numerous vacuoles and secreted electron dense discoidal granules of varing size. At the base of integument there is a basement membrane which forms a demarcation with the muscle layer under integument. There are the fiber layer which constructed from net-work of collagen like fiber between the basement membrane and muscle layer. The nucleated integumental cell, which is called a epithel cell, is situated in the deeply parenchyma. The protoplasmic tubule, which originates from the base of the integument, piercing through the basement membrane at various points, and connected the integument and deep interior epithel cell each other. The epithel cell has one or more nucleus, and electron dense secreted granules, mitochondria, endplasmic reticulum and Golgi complex were observed around nucleus. Then, the body wall is a large syncytium constructed from the anucleated integument and nucleated integumental cell. Underneath the integument with the basement membrane as the boundary, there are such muscle layers as longitudinal, circural, oblique and dorso-ventral muscle layers that are arranged relatively nearly. Myofilaments, like the muscle in other parasite, can be classified into two kinds of thick and thin. The paranchyma at the deep region underneath or between the muscle cell has numelous vacuoles of which lock like sponge structure while others are electron dense contents. And mitochondria are relatively scarce, and flame cells can be observed in some places. The digestive canal is made up from the single layered intestinal epithel and thin muscle layer. The numerous but relatively short microvilli, high densegra- nules, endoplasmic reticulum and mitochondria are observed in the intestinal epithel cell. Yolk gland is the same as that in other Trematoda, with dense granules of varing size around its nucleus, and with well developed endplasmic reticulum as well. The ovary was covered with relatively thick outer membrane. The germ 94

cells which were almost round, and in the ovary there are vacuoles of varing size here and there, and small dense granules are lined up almost along the cell membrane. The many vacuoles of various size were observed in numerous germ cells. Nuclei were large in size, and had the double membrane structure. The large nucleoli in these nuclei can be observerved. Many mitochondriae with unclean cristae can be observed around the nucleus. Numerous spermatozoa are found in the testis in the process of their formation, and the spermatozoa resemble closely to those in other Trematoda. The exfoliation of integument is observed in relatively early chainging after treated with ambilhar.

2. Clinical Observations on the Treatment of Schistosomiasis japonica with Nitrothiazol Derivative Ciba 32 644Ba

Makoto Kurata, Ryotoku Nishimura, Shoji Nagaoka, Masaaki Yoshizumi, Masaaki Koga, Yoichi Kojima, Toshihiro Imamura, Shoji Nomori, Haruhiko Tsuda, Hakun Inoue and Teruyoshi Yamamoto The First Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan

Ciba 32 644 Ba which is a derivative of nitrothiazol was administered for seven days to 36 patients of Schistosomiasis japonica (4 of acute type and 32 of chronic type) dwelling in endemic areas in the ChIkugo basin. The dosage applied was in a ratio of 20 mg/kg of body weight/day. Clinical progresses of these patients were surveyed before, immediately after, 3 months after, and a year after the administration. Results: In the cases of acute schistosomiasis, fever (the fever was lowered to normal by this drug in all cases of acute type) and eggs in feces disappeared 3 months or a year after the treatment, liver functions examined with Z. T. T. and T. T. T. recovered to near normal level 3 months after, the increase in gamma globulin in serum protein fractionation, which had been observed in 4 cases of acute schistosomiasis, began to decrease 3 months after, and the slight increase in transaminase value, which was observed in 13 of 33 cases, returned to a normal value at the third month of the treatment. There was no change in LDH due to the treatment. Although all was slightly increased before the treatment, no fluctuation in its value was observed immediately after, 3 months after and a year after the treatment. There was no remarkable change in serum cholesterol. In the examination of peripheral blood, leucocytoses (above 10,000/mm3) was observed in 7 of 36 cases, but the white cell number of these patients was recovered to near a normal value 3 months after, except 2 cases of acute type. Although acidophilia was seen in 9 (4 of acute type and 5 of chronic type) of 36 cases, the case number was reduced to 5 (3 of acute type and 2 of chronic type) 3 months after the treatment, and all cases, except for one case of acute type, showed normal hema- tologic figure a year after. As side effects, headache was occurred in almost all cases, nausea, anorexia, 95 feeling of fatigue, etc. were present in some cases, and the change in ST and T (lowering) in ECG was observed in about 20 %. When the dosage was yarned as 15, 20 and 25 mg/kg/day, 20 mg/kg/day was supposed to be most adequate from the viewpoints of side effect and therapeutic efficacy.

3. Interdermal Test for Microzome Fraction of Schistosoma japonicum

Kogo Okabe, Yuzuru Tomooka and Minoru Akusawa Department of Parasitology, Kurume University School of Medicine, Kurume, Japan

Rough and Smooth encoplasmic reticulum fractions of Schistosoma japonicum were prepared with ultra centrifugation by Houle method. Each of the fractions was used for the antigen of interdermal test and compared with Melcher antigen.

4. Further Studies on the Laboratory Breeding of Oncomelania Snails, the Intermediate Host of Schistosoma japonicum

Hajime Matsuda, Junji Kobayashi and Manabu Soso Department of Parasitology, Institute of Medicine, University of Tokyo, Tokyo, Japan

Because of difficulties in the laboratory breeding of the snail intermediate hosts, experimental studies with Schistosoma japonicum have remained far behind the progresses in those on S. mansoni. The authors carried out a series of studies on the methods for laboratory breeding of Oncomelania snails under various conditions, and a standard technique has been established, with which 0. nosophora from Yamanashi was successfully bred to the fifth generations, 0. nosophora from Kurume to the third, 0. quadrasi from the Philippines to the third, and O. formo- sana from Taiwan to the third generations. Two kinds of containers were used according to the developmental stage of the snails. Sexually mature snails were transferred into unglazed potteries 20 cm in diameter and 5 cm in height containing wet soil in the bottom, and were kept for about a month until they deposit sufficient numbers of eggs. The newly hatched young snails were transferred to standard tropical fish aquaria (glass tubs, 30 cm long, 20 cm wide and 25 cm high) containing soil and water to a depth of about 10 cm. An airpump filter was placed in the soil of each aquarium to keek the water always clean. A fluorescent lamp (puple light) was placed on each aquarium. Powder of laboratory animal food (CLEA, for mouse and rat) was provided as nutrient for the young snails. Increase in the speed of growth was seen in containers to which pieces of rice straw, cal- cium carbonate or oyster shell powder was further added. Newly hatched snails transferred to the aquaria developed to average shell length of 5.6 mm in one 96 month, and began to lay eggs after two months when they reached to the average shell length of 7.1 mm.

5. Schistozomiasis haematobia in Congo

Toshio Yamamoto, Hiroshi Takahashi, Hiroyuki Amano, Toru Yamanaka and Masanori Aihara Tenri Hospital, Tenri, Miie, Japan

Twenty-one cases of the Schistozomiasis haemotobia infestitution were treated in our clinic in Congo from October, 1967 to May, 1968. The patients with the schistozomiasis haematobia infestation were character- istically distributed along the Koulou River especially in Brazzaville and Jacob city . On urinalysis ova were found in all cases, and proteinuria, haematuria (microscopic), urobilinogenuria are 81, 71 and 52 % respectively. CBC revealed no specific abnormalities except for rare occurrence of eosinophilia. Sodium Antimonyl tartarate was proved to be the drug of choice for the treatment of Schistozomiasis infestitution.

6. The in vitro Cultivation of Metagonimus yokogawai from the Metacercaria to the Adult Stage

Kazuo Yasuraoka, Kuniko Kojima and Yoko Takemura Department of Parasitology, N. I. H., Tokyo, Japon

Metagonimus yokogawai has been successfully cultured in cell-free media under axenic conditions and an air phase of 5 % CO2 in air from the metacercaria to the adult stage. Sexually mature worms containing eggs without shells were morphologically normal, although somewhat stunted in growth. It has taken at least 3 to 4 times longer for adult worms to develop in culture, than in the host.

7. Achillurbainia sp. found from Rats in Malaysia (Trematoda : Achillurbainiidae)

Ichiro Miyazaki* and Eh Hoa Kwo** *Department of Parasitology , Kyushu University, Fukuoka, Japan and **Institute or Medical Research, Kuala Lumpur, Malaysia

During our survey on the lung fluke in Malaysia, we found in 1968 Parago- nimus-like eggs in the feces of rats, Rattus muelleri living along the stream in the forest. Dissecting them, we obtained mature flukes from their trachea, which were identified as Achillurbainia sp. Since 1939, when A. nouveli Dollf us were 97

reported from an eyelid abscess of a leopard in Malaysia, the mature fluke of the same genus was found from the maxillary sinus of an opossum in Brazil and from a retro-auricular abscess of a girl in China. In addition, a mature fluke or eggs of the closest genus Poikilorchis belonging to the same family Achillurbainiidae were reported from subcutaneous abscess of humans in Africa and Borneo. The habitat observed in these cases was purely accidental. The natural final host and the natural habitat of Achillurbainia sp. were first clarified by our investigation.

GROUP G : Medical Entomology

1. Fine Structure of Aedes aegypti

Seiichi Inatomi, Daigoro Sakumoto, Yasumasa Tongu, Setsuo Suguri and Kazuo Itano Department of Parasitology, Okayama University Medical School, Okayama, Japan

The purpose of this study is to obtain the normal pattern of the fine structure of digestive system of Aedes aegypti.

Method The collected tissues from living mosquitoes were fixed with phosphate buffered 1 % cold gultaraldehyde solution (pH 7.4) for 30 minutes and were washed well with phosphate buffer. After that, they were post fixed for 2 hours in phosphate buffered cold 2 % osmium tetroxid solution and embedded in Epon 812 after dehydration with ethanol series. The spacements after sectioned with Porter-Blum ultramicrotome were stained with uranyl acetate and lead nitrate.

Result This report deals the fine structure of Aedes aegypti with of the digestive system. The glandular cells observable in the salivary gland have a very unique morphology ; namly, their nuclei as well as mitochondria and other organellae are practically all forced to one side, assuming a flate shape, and the major portion of the cell is filled with minute granules of low density. Many such glandular cells are aggregated, forming a multicellular gland. Next, in the digestive canal, numerous microvilli are observed growing densely on the inner surface of mid-gut, but no such microvilli can be seen in the for-gut and hind-gut. Near the roots of these microvilli there are numerous mitochondria. From the junction of the mid-gut and hind-gut, five Malpigian tubules extend outward, and the Malpigian lumens open into the digestive canal at this junction. The two or three epithel cells are distributed on the wall of Malpigian tubule in cross section. The three layers which are spinal, intermediate and basal zones, can be observed in this epithel cell layer. The numerous mitochondria and endplasmic reticulums at the near part of the 98

apical zone. There are many varing size of lime-granules of which diameter is 0.1 to 3 microns, and vacuoles in the intermediate zone. These granules have 2 to 7 layers. The basal zone of these cells cotains an elaborated infolding of the cell membrane, which is called a basal striation. There are also countless numbers of microvilli growing densely on the inner surface of the Malpigian tubules, and usually these microvilli are filled with char- acteristically shaped mitochondria, some of which are differing and extended to the two or three microvilli. At the end of digestive system, the rectum covered with thin culticule at the inner surface of the rectum lumen, has the rectal gland (papillae) having several glandular cells with numerous endplasmic reticulum, as associated organ.

2. Studies on Biology of the Culex tritaeniorhynchus Group, the Principal Vector of Japanese Encephalitis

Yoshitake Wada*, Mamoru Watanabe*, Manabu Sasa* and Seiichi Inatomi** *Department of Parasitology , the Institute of Medical Science, the University of Tokyo, Tokyo, Japan, and **Department of Parasitology , Okayama University Faculty of Medicine, Okayama, Japan

A series of field studies were carried out on biology of mosquitos of the Culex tritaeniorhynchus group were carried out in several stations in Okaykma Prefecture in 1967 and 1968. Investigations were made on the seasonal distribution of the population density of adults (collected by light traps, dry ice traps and by biting collections on pigs and humans) and larvae, and the egg composition of the females. Their nocturnal biting rhythms were observed also by the pig bait collections, in which both C. tritaeniorhynchus summorosus and C. pseudovishnui showed roughly two peaks in a night. Studies were made also on the resting behavior of the adults. Both males and all gonotrophic stages of the females were found to be resuting during the daytime in mainly bushes of low and flat leave plants, such as sweet potato and strawberry, became active after sunset, and the females frequently take temporary rests on trees and walls before and after the engorge- ment. Details of these studies are published in Jap. J. Experimental Medicine vol. 38, No. 4.

3. Laboratory Colonization of Culex Tritaeniorhynchus summorosus

Akiko Shirasaka, Yoshitake Wada and Manabu sasa Department of Parasitology, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan

The first successful results on the laboratory colonization of the mosquito C. tritaeniorhynchus summorosus were obtained by Newson and others (1956) with a 99 mixture of larvae from Honshu, Kyushu and Okinawa as the starting material. The use of pigs as the host for the blood meal, especially designed large mosquito cages, and application of long day artificial illumination were considered as require- ments for breeding of this colony. However, progeny of the same colony provided to us in 1965 was shown to be easily bred in 30 mm3 cages, by feeding on laboratory mouse and without providing artificial long day illuminations. Trials for colonizing the same species of mosquito collected from areas near Tokyo have been made repeatedly during the past several years, but were all unsuccessful because the laboratory reared males and females of the second generation did not copulate in the cages. However, two colonies of the mosquito collected from Okayama in 1967 were able to produce a few fertilized egg rafts at the second generation, and the offsprings could be bred for the succesive generations in our laboratory using the small 30 mm3 cages. The hatching rate of the egg rafts gradually increased according to the generations, such as 25.0 % (7 or 28) at the second, 36.4 % (4 of 11) at the third, 38.9 % (7 of 18) at the fourth, 42.9 % (21 of 49) at the fifth, and further to 93.3 % (42 of 45) at the tenth generation. It has further been clarified that the already established three laboratory colonies differ greatly in biological characters from the wild colonies, in that they copulate in the cages at high percentages, and are able to produce fertilized egg rafts easily in the laboratory. Such characters are considered to be genetically fixed, and have been established from the wild colonies through environmental selections under the laboratory conditions. At hybridization experiments between the wild and the laboratory adapted colonies, the results have been always con- sistent in that the crosses of the wild males and the laboratory adapted females could efficicntly copulate in the cages and produced fertilized egg rafts, while the egg rafts obtained at the reciprocal crosses between laboratory adapted males and wild females were almost unexceptionally infertile, thus showing that such steno- gamous character of the laboratory adapted colonies was associated mainly to the females. On the other hand, the reproductivity in the hybridization experiments between some East and West Pakistan laboratory strains of C. iritaeniorhynchus tritaeniorhynchus and the Japanese strains of C. t. summorosus were all excellent, and there seemed to exist no incompatibility between the two " so called " subspecies.

4. Salivary Chromosomes of Culex pipiens fatigans and Some other Species of mosquito

Tozo Kanda* and Genzo Mitsui** *Department of Parasitology Institute of Medical Science, University of Tokyo and **Japan Associationfor Tropical Medicine, Tokyo, Japan

Salivary chromosomes of five species were observed, i. e. Culex pipiens fatigans, Wyomia smithii, Anopheles sinensis, A. sineroides and Anopheles sp. from Thailand. Chromosome maps were prepared with relative ease in Genus Anopheles and Wyomia. Chromosomes of these genus are fairly short and they spread well. 100

With Culex, however, the problem of preparation was more difficult. The reasons might be as follows : a) The chromosomes are much longer and do not spread well. b) The chromosomes tend to stick together. c) There are some abnormal figures of chromosomes. Thus, to facilitate the preparation technique, selections of strains which have many good preaprations were continued by using sublima- tiong methods and time point of dissection was found. The best figures resulted from dissection during the optimal time period which is 6 to 12 houres age of the 4th inster larva at a temperature of 17-18°C. Detailed maps have been made of the salivary chromosomes of Culex pipiens fatigans. The chromosomes appear as long, paired, synaptic, polytene chromosomes with characteristic banding patterns. Each nucleus contains three pairs of these synapsed chromosomes, each pair distinguishable by length, ratio of the arms, and by banding patterns. There is no definite chromocenter. Unlike Anopheles in which the chromosomes are united in a chromocenter, the choromosomes of Culex spread, individually. There is a differentiated swollen region which serves as a point to separate the right and left arms of each chromosome. Using these characteristic in the form bulbular regions without definite band as markers, each chromosome can be identified. These should occur the centromere. The banding pattern in all chromosomes seems to be strongly similar between Japanese K-Strain and Malaysian PJ-Strain. In the comparison with the mape of Culex pipiens by Laven there are many different areas of banding pattern especially in the left arm of chromosome 1. The salivary complements of A. sinensis, A. sineroides, A. atropalvus and Ano- pheles sp. from Thailand show the same general pattern, consisting of three pairs of closely synapsed polytene chromosomes with reproducible banding patterns. There are many gross and some detailed resembrance to the chromosomes of atroparvus and therefore the various chromosomal arms have been designated in a maner similar to those of atroparvus of which detailed maps were already made by Frizzi. Thus the shortest chromosome which appears as a telocentric rod has been designated the X, the autosome whose arms of about equal lengths has been called chromosome 2, and the autosome with unequal arm lengths chromosome 3. These maps have made possible the study of simiarities and differences in the banding patterns of these species. Detailed maps have been made of the salivary chromosomes of Anopheles sinensis and A. sineroides. In the comparison of these two species there is a strong similarity in the banding patterns of chromosome 2 and 3, but there are some different banding patterns in X chromosome, though these are taxonomically very close species. Detailed maps have also been made of the salivary chromosome of Wyomia smithii. The chromosomes appear as relative long, paired, synaptic, polytene chromosomes with characteristic banding patterns. Each nucleus contains three pairs of these synapsed chromosomes, each pair distinguishable by length and by banding patterns. There is strong similarity in the figuar of bulbular regions without definite chromocenter, between Culex pipiens fatigans and this species. 101

5. Survey of Ticks in Thailand and their Life Cycle

Yutaka Saito *, Chamlong Harinasuta** and Cherdlarp Vasuvat** *Department of Medical Zoology, Niigata University School of Medicine, Niigata, Japan, and ** Faculty of Tropical Medicine, University of Medical Sciences, Bangkok, Thailand

During the past one year from August 1967 to July 1968, a survey of ticks was made mainly in Nakornratchasima Province and partly in others of Thailand, using the techniques of flagging, direct observation of tree leaves and host examination. The area surveyed included open plain and jungle terrain, and the following results were obtained. Twenty-seven thousand and seventy-seven ticks in all, including both adults and immatures, were collected by the above methods and classified into 8 genera and 33 species. They were members of the genera Ixodes (1 sp.), Haemaphysalis (18 spp.), Rhipicephalus (2 spp.), Boophilus (1 sp.), Dermacentor (4 spp.), Amblyom- ma (3 spp.), Aponomma (3 spp.), and Nosomma (1 sp.) as shown in the list and summarized data of the collection. Most of these ticks are demonstrated on the table. On the other hand, the life history of 17 tick species (Ixodes granulatus; Haemaphysalis bandicota, H. bispinosa, H. cornigera shimoga, H. formosensis, H. lahrangei, H. leachii subsp., H. obesa, H. p. kinneari, H. p. papuana, H. wellingtoni ; Rhipicephalus s. sanguineus, R. bequaerti; Boophilus microplus; Dermacentor aura- tus ; Amblyomma javanense and Amb. testudinarium) has been observed by rearing in the laboratory, in which normal rabbits, white rats, mice and chickens were used as experimental hosts of these ticks. The rearing results are indicated in the table. From the course of the rearing, the immature forms that had been still undescribed and the biology of most Thai ticks have become evidenced.

6. Genetical Linkage Relationships of a Malathion-resistance Factor in the Culex pipiens Group

Takeo Tadano Department of Parasitology, the Institute of Medical Science, the University of Tokyo, Tokyo, Japan

Several mutants have become available for informal genetics of insecticide- resistance in this species. The purpose of this experiment is to investigate the chromosomal relationship of a malathion-resistance gene in Culex p. pallens collected in Hyogo Prefecture, Japan, by crossing with two marker strains, ru (ruby eye) and y (yellow larva) of C. p. molestus. The results of crosses and backcrosses between the malathion-susceptible marker strains (0.035-0.04 ppm) and malathion-resistant strains (LC50 0.9 ppm) showed 102 that the malathion-resistance in this species was due to a single incompletely dominant gene which is linked to ru on chromosome 2 with crossover units of 40 % in the males and 43 % in the females. From the crossing with y strain it is considered that the gene sequence on chromosome 2 is y-ru-mr (mr indicates a gene for malathion-resistance)

7. Insecticide Resistance to Organophosphorus Compounds in Culex Pipiens S. L. Larvae

Takeshi Suzuki* and Tokuko Umino** * Division of Pest Control, Japan Environmental Sanitation Center and ** Department of Parasitology, Institute of Medical Science

A nearly pure resistant colony of Culex pipiens to malathion or to diazinon was established by selection with each chemical. A nearly pure susceptible colony was also established by " discriminating method " from a mixed population. The genetic mode of the resistance to malathion or to diazinon was proved to be monofactorial with incomplete dominance of the resistant gene. In 1966, a multiple-resistant or -tolerant colony of Culex pipiens pallens toO-P compounds was found in the Central Honshu of Japan. After selection for 12 generations under the pressure of fenthion, the resistance raised not only to fenthion but also to almost all of O-P compounds. The separation with fenthion for 12 generations by the " discriminating method " could established a nearly sus-. ceptible colony to fenthion, which was also susceptible to several other O-P compounds.

GROUP H: Venom and Poisonous Snakes

1. Studies on the Immuno-Electrophoretic Separation of Habu Venom and It's Detoxication in vitro

Hachiro Sato*, Masao Yogi*, Masami Kazama* and Shigeo Takaki** * Second Department of Internal Medicine, Faculty of Medicine, Kagoshima University, and ** Department of Internal Medicine, Kagoshima Communications Hospital, Kagoshima, Japan

The comparison between the venom antivenin arcs and the pattern of normal human serum was made in order to identify the immunological nature of protein moiety of the venom. 103

The effect of some chemicals upon proteolytic activity of the venom, the reducing effect of some chemicals on cytotoxicity of tne venon against HeLa cells, and the correlations between the cytotoxic effects of principal chromatographic fractions of Habu venom on HeLa cells and the proteolytic activity were also investigated. Method Venom : crude venom of Habu, Starch gel electrophoresis : Smithies method Immunoelectrophoresis : observed after 26 hrs Proteolytic activity : phosphate buffer at pH 7.5 with casein as substrate Cell culture : HeLa S3, Fagle's minimal essential medium (1959), replicate culture, observed by nucleus enumeration or by morphological change Chemicals : MPG (a-mercaptopropionyl glycine), Trasylol, Glutathione, a-Lipoic acid CM cellulose column chromatography : 19.5 X 1.8 cm, acetate buffer pH 5.8, stepwise from 0.005 M to 0.5 M Result and Conclusion 1) Seven fractions were identified by zone electrophoresis. 2) Twelve precipitin arcs were verified in venom-antivenin immunoelectrophoresis. 3) MPG was found to be very inhibitory to proteolytic activity of the crude venom. On the contrary, Trasylol had no intended effect. 4) SH-compounds such as MPG, glutathione and a-lipoic acid showed their noted reducing effects on cytotoxicity of the venom against HeLa cells. 5) Tubs No. 80 of the highest proteolytic activity produced a remarkable rounding of HeLa cells. Tube No. 4, however, gave rise to pyknosis, plasmolysis or cell detachment from the glass surface. Rounding of the cells was not notice- ably remarkable.

2. A Study on the Prophylactic Toxoid Against Venom of Certain Asian Snakes

Yoshio Sawai*, Yoshiharu Kawamura* and C. S. Tseng** * Institute of Medical Science, University of Tokyo and ** NAMRU-2, Taipei, Taiwan

The purpose of this study is to help the medical treatment of severe poisonous snake-bite, because there would be a limit in serum treatment of patient who received a large amount of venom. In previous reports, Sawai et al. showed that antigenicity of habu venom (Trimeresurus flavoviridis) which was inaectivated by dihydrothioctic acid (DHTA) were tested for experimental animals, and a large scale of field trials of prophylactic immunization were started in Amami and Ryukyu Islands of Japan from 1965. The program is now being continued. 104

This report concerns an attempt to immunize experimental animals with five kinds of Taiwanese snake venom, Trimeresurus mucrosquamatus, T. stejnegri, Agkistrodon acutus, Bungarus multicinctus and Naja n. atra and one kind of venom of Ryukyuan snake, Trimeresurus elegans which were inactivated by DHTA. Those immunized animals were challenged intramuscularly by each kind of untreated venom for the test of antigenicity. Blood levels of antibody of immunized animals were also investigated. The results indicated that the antigenicity of the venom of T mucrosquamatus, T. stejnegri and T. elegans seems to be closely related, and rabbits which were immunized with one of the three kinds of venom showed cross-protection against local effect with each other. Polyvalent toxoid of T. mucrosquamatus, T. stejnegri and A. acutus is good antigenic against local effects of venom of each other. Mice which were immunized with venom of B. multicinetus which was treated by DHTA showed good anti-lethal effects. More amount of DHTA is neccessary to inactivate venom of Naja n atra. And the antigenicity of the toxoid seemed not as good as that of B. multicinctus.

3. A Study of Snake-Bites on Taiwan

Yoshio Sawai* and C. S. Tseng** * Institute for Medical Science, University of Tokyo and ** NAMRU-2, Taipei, Taiwan

There is no reliable report on snake-bites on Taiwan after the War. The purpose of this study is to elucidate the physical condition of snake-bites and to make clinical analysis of them on Taiwan and to contribute to the improvement of medical treatment and prophylaxis of snake-bites. 891 cases of poisonous snake-bites in five years from 1964 to 1968 were recorded. Snake-bites by Trimerusurus mucrosquamatus, Trimeresurus stejnegri and Bungarus multicinctus were occurred in common, and those by Naja naja atra and Agkistrodon acutus were frequently occurred in South Areas, and snake-bites by Vipera russelli restricted in East Area. In 891 cases, 19 patients died of bites by T. mucrosquamatus, Bungarus multicinctus and A. acutus, and 46 patients had motor disturbances after the recovery from bites by T. mucrosquamatus and Naja n. atra. 105

GROUP M: Miscellaneons Tropical Diseases

1. Five-year Survey of Vibrio parahaemolyticus in Foreign Waters by Ocean Navigation ; A Summary

Yoshio Aoki Department of Bacteriology, Nagasaki University School of Medicine, Nagasaki, Japan

From a large number of sea-water, plankton, marine-mud, and fish samples collected in Southeast Asia and Central Pacific during the period 1964-68, 420 Vibrio parahaemolyticus strains were isolated, among them 382 strains were K- typable within the scope of K1 - 32. The results of K-typing were not similar to those of strains isolated from food-poisoning cases in Japan, and bear rather some resemblance to those of strains originated from sea-water and marine products collected in Japanese waters. The same could be said of Kanagawa phenomenon, a newly advocated characteristic related to the hemolytic activity of human strains ; only 10 among the total were positive to this test. To make a thorough investigation for the presence of food-poisoning due to V. parahaemolyticus and for healthy carriers of this organism in foreign countries especially in the tropical and sub- tropical zone, is considered to be the key to on understanding of epidemiology of this type food-poisoning.

2. Possibility of Treatment of Sickle Cell Anemia in Congo

Toshio Yamamoto, Hiroshi Takahashi, Hiroyuki Amano, Toru Yamanaka and Masanori Aihara Tenri Hospital, Tenri, Mile, Japan

Eighteen sickle cell disorders (sickle cell anemia and sickle cell traits) were treated from October 1967 to May, 1968, in Congo. These were diagnosed by bisulfite method, clinical symptom and routine hematological tests. Many difficulties on therapy were not infrequently encountered during the course of observation of sickle cell anemia. However our experience in Congo suggested the possible available method for the symptomatic improvement. 106

3. Observation of Several Interest Diseases in Ie Island, Okinawa

Shigeo Iwata*, Tsuneji Araki*, Moriyoshi Chuda*, Toru Fujihira*, Kazuo Ikoma*, Minoru Yasuhara** and Teizo Hashimoto** * The 2nd Department of Internal Medicine, Osaka Medical College, Osaka, Japan, and ** Department of Dermatology, Osaka Medical College, Osaka, Japan

Purpose & Method : In the summer of 1967, the authors performed examinations of internal medicine, parasitology and dermatology on Ie Island, Okinawa. For internal medicine a complete physical examination including urinalysis was performed. For parasites the stool was examined by means of thick smear technique with cellophane cover and especially for pinworm cellophane-type method 2 times was used. For dermatological data we performed by means of inspection, palpation and histological examination. Result : Of the 1398 subjects (508 male, 890 female) examined medically, there were many urobilinogen positive patients. Blood pressure was lower than subjects in Japan. Subjective symptoms were headache, vertigo, fatigue, shoulder pain, palpitation etc. In the diagnosis of cardio-vascular system 131 (9.4 %) had hyper- tention, 53 (3.8 %) had hypotention, 36 (2.6 %) had functional and 28 (2.0 %) organic heart murmurs. Of the digestive diseases 145 (10.4 %) showed liver disease including hepatomegaly, 29 (2.1 %) had gastritis. Of the urological diseases 52 (3.7 %) had albuminuria, 13 (2.6 %) patients with were found with hydrocele, 9 (1.8 %) patients with swelling of the testicles. Of the blood diseases 128 (8.9 %) had anemia. In 2672 parasitic examinations 168 ovapositive cases (6.3 %) were found. Of these 125 (4.7 %) were hookworm infections. Of the 2230 subjects examined by the cellophan-tape method 2 times, 178 subjects were found to be positive for pinworm ova. There were more positive cases in the younger age group. In the 294 subjects (110 male, 184 female) of dermatological examination, 81 (27.6 %) were found to have pyoderma, 77 (26.2 %) enzyme-dermatitis group, 66 (22.5 %) sweet gland diseases. Conclusion : Many cases of liver diseases, anemia and hypotention are thought to be attributed to malnutrition. Representative skin disease observed on Ie Island were as follows : (1) Ecthyma vulgare (6.8 %), (2) Contact dermatitis due to " Zori " (11.9 %), (3) Chronic polymorphous light eruptions (4.8 %), (4) Elephan- tiasis, chronic orchitis and hydrocele caused by possible epidemic filariasis (4.4 %). 107

4. Valid Trial for Mental Health on Practical Use Among Malaysian Students

(1) A study in Sabah State

J. E. B. Ambrose*, Kimio Yoshikawa** and Naomi Kodaka** * Director Teacher Training Ministry of Education, Malaysia, and ** Department of Biology, Faculty of Science Osaka City University, Osaka Japan

There are many projective test for personality analysis in the world and we adopted Koch's Baumtest (tree-test) for students. According Koch (1952) three is a relationship between tree form and human form, that plant life in the tree, as in a standing status, reaches the highest resemblance to humanity. This test is one of the most simple for psychodiagnosis and is showing its direct response of results against Rorschach test or TAT is rather passive one, and can be use for anyone from kindergartner to adult. In this time we applied this test to diagnose for mental health of students in Sabah, Malaysian Borneo. As the results of this test, we can found the same analogy among lower school children in Europe, Japan and Sabah. This means that the personality in infancy actually depends upon their innate behaviour. So we can see an approach to the study of human ethology in this test. After childhood, individuality and personality including mental health is formed gradually under their own cultural condition. In puberty, there can be seen an influence of abundance of vegetal nature to individuality and personality in Malay- sian than Japanese, and also we can assume that there are some differences among races in Malaysians. On this fact this method can be applied to comparative study on cultural- and ethno- anthropology. Generally speaking, mental health is related to their physical health. Especial- ly, many disease, that is ptosis, peptic ulcer, heart neurosis, etc., are caused by mental disorder which is concerned with autonomous (vegetative) nervous system through stress. We are wishing to research on a field between physiology and behaviour which is effected by coaction of body and mind in cultural condition. It seems to be that there are some possibility in diagnosis of this field through that Baumtest.