Vol. 4 No. 4 171 December, 1963 LABORATORY MEETING held under the auspices of the ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE on June 22, 1963 at the PARASITOLOGY DEPARTMENT, UNIVERSITY OF SINGAPORE.

Dr. Don E. Eyles, Dr. A. A. Sandosham, Dr. M. Warren, Dr. S. P. Ramakrishnan, Miss Elizabeth Guinn and Mr. Yap Loy Fong A POSSIBLE NEW MONKEY MALARIA PARASITE FROM THE NILGIRIS IN SOUTHERN INDIA

Studies have been conducted of a parasite iso- The older asexual stages are quite abundant lated from Macaca radiata in the Nilgiris, Ma- in the peripheral blood, but there does appear dras State, Southern India (Ramakrishnan and to be a distinct tendency for the parasites to re- cede from the blood as in P. coatneyi Mohan, Bull. Nat. Soc. India, Med. Mosq. Dis., peripheral and P. knowlesi (particularly the new subspecies 9: 2, 1961). The characteristics differ from those of Garnham). The closest affinities would appear of previously described parasites. to be with these two parasites. The periodicity appears to be tertian, but more Studies of the invasion of reticulocytes show studies need to be done on this important point. that this parasite does not prefer the young ery- The ring stages are similar to those of P. coat- throcytes. In contrast, the young trophozoites of neyi and also P. knowlesi and P. falciparum. The P. coatneyi show a marked predilection for the reticulocytes. Should the periodicity be demon- older trophozoites are extremely dense and com- strated with certainty to be tertian, there would pact and have the most abundant pigment seen appear little doubt that the parasite is undes- by us in any monkey malaria parasite. The schi- cribed. Should it later prove to be quotidian zont appears to have about 15-19 merozoites. (which we believe is very unlikely) comparison Distortion and a pinkish discoloration or vague must be made with the subspecies of P. knowlesi Schuffner type stippling of the erythrocyte are (the description of this subspecies is not in press, produced. Maurer clefts are not seen. The ga- so its name cannot be used at this time). metocytes are round and have very heavy pig- Microscopical preparations showing the va- ment, sometimes clumped in a few large masses. rious erythrocytic stages were demonstrated.

Mr. Yap Loy Fong and Dr. A. A. Sandosham BABESIA DECUMANI (MACFIE) AND OTHER BLOOD PARASITES IN A LITTLE SPINY -RAT, RATTUS MUSSCHENBROEKI (=R. WHITEHEADI)

Blood parasites identified as Babesia decu- cording to the records kept at the Malaria and mani (Macfie) by Marshall Laird (personal com- Filariasis Research Division of this Institute (see munication) were found in three giant long-tail- Table below) there was an infected R. sabanus ed rats (Rawls sabanus) collected at the Nation- in 1957 from the Ampang Reservoir Forest Re- al Park in north Pahang by the Institute for serve in Selangor. in Medical Research 1955; six years later two The present finding of B. decumani in a Raï - further rats (R. sabanus and R. bowersi) from tus musschenbroeki (=R. whiteheadi) from Ba - the same area were reported harbouring Babesia tang Berjuntai, Selangor indicates another new parasites presumably of the same species. Ac- host and a different locality. 172 SINGAPORE MEDICAL JOURNAL

FOREST RATS FOUND INFECTED WITH BABESIA DECUMANI (MACFIE).

Species (No. infected) Locality Year

Battus sabanus (3) National Park, Pahang 1955 (I) Ampang Reservoir Forest Reserve, Selangor 1957 (I) National Park, Pahang 1961

Rattus bowersi (I) Pe Rattus musschenbroeki (=R. whiteheadi) (1) Batang Berjuntai, Selangor 1963

It is interesting to note that all the rats found and the flagellum extends along the broad un- with natural Babesia infections hitherto in Ma- dulating membrane and beyond the anterior end laya belong to the forest species. the free portion measuring about 7 g. The Giemsa-stained thin blood film showed The thin film also showed unidentified micro- the parasites of B. decumani mostly as vacuolat- filariae measuring about 118-163 g long and 2-3 ed ring -shaped unpigmented bodies which are g broad. The purple -stained nuclei are large and not unlike Plasmodium falciparum, measuring closely packed. The tail ends in a large rounded 2-4 µ in diameter, and a few amoeboid forms nucleus which gives it a clubbed appearance. with red nuclear chromatin and scanty pale blue The blood film was demonstrated together cytoplasm in unenlarged erythrocytes. with drawings made from the microscope of Basic stained bacilliform organisms, 7-24 of these blood parasites. them measuring about 1-1.7 g in length and 0.3 REFERENCES in width were seen in a few erythrocytes. They resemble microti Lavier. Baker, J.R., Chitty, D. and Phipps, E. (1963). Para- Grahamella sitology, 53, 297-301. Also found in the same blood film was a Try- Hoare, C.A. (1949). Handbook of Medical Protozoo- panosoma sp. belonging to the lewisi group as logy. Ballière, Tindall and Cox, London. classified by Hoare (1949). It is about 35 g long Institute for Medical Research, Kuala Lumpur. An- nual Report for 1961, p. 86. with a maximum breadth of 7 g. The posterior Jaffe, J.J. and Doremus, H.M. (1963). Nature, Lond., two-thirds of the cytoplasm stains an intense 198, 556-558. blue. The nucleus is large (2.3 g) and rounded Lavier, G. (1921). Bull. Soc. Path. expt., 14, 569-576. and is situated at about the middle of the body. MacFie, J.W.S. (1915). Ann. trop. Med. Hag., 9, 457- The kinetoplast is about 1.5 g behind the nucleus 494.

Dr. Gordon F. Bennett, Dr. Don E. Eyles, Dr. McWilson Warren and W. H. Cheong PLASMODIUM JUXTANUCLEARE (?), A NEWLY DISCOVERED PARASITE OF DOMESTIC FOWL IN MALAYA

Plasmodium juxtanucleare was first described cribed for P. juxtanucleare has led us to term by Versiani and Gomes in 1941 from galliforms provisionally this parasite P. juxtanucleare. A in Brazil. Recently, Dhanapala (1962) described survey of the chickens from the same region this parasite from galliforms in Ceylon. In a re- from where the infected mosquito was obtained cent survey by this laboratory of culicine mos- showed that nine of them were infected with the quitoes of a coastal mangrove region near Kuala same parasite. Three of 20 chickens in the IMR Lumpur, sporozoites from a naturally infected grounds were also infected naturally with the Culex sitiens produced a Plasmodium infection parasite. when injected into a domestic chick. Subsequent- ly this parasite was transmitted by (i) blood ino- culation and (ii) sporozoites from experimental- CYCLE IN THE FOWL ly infected Culex sitiens. The similarity of the Studies in progress in the Malayan P. juxtanu- erythrocytic stages of this parasite to those des- cleare indicate a prepatent period of 14 days, a DECEMBER, 1963 173 high parasitaemia for 18-28 days and a long entiation of sporozoites within the peduncle has period of chronicity; the original infection still not been observed. Following the release of the showing parasites some 22 months following sporozoites and the disintegration of the oocyst, infection. Transfer of the parasites by blood ino- the peduncle persists for some time. Apparently culation has given variable results-all birds the tissue in the peduncle differs from that of the have become infected at times varying from 1-15 oocyst; it has not yet been determined whether days following inoculation of infected blood. a septum divides the peduncle from the oocyst. Birds inoculated with blood intravenously be- came infected more quickly than those inocula- The erythrocytic stages, oocysts and sporo- ted subcutaneously. zoites of Plasmodium juxtanucleare were de- monstrated. In addition, sporozoites of Plasmo- CYCLE IN CULEX SITIENS dium traguli and Plasmodium cynomolgi in Anopheles umbrosus and Anopheles maculatus The parasite requires a minimum of 13.5 days respectively were demonstrated in a comparison to produce sporozoites and the oocysts do not of different sporozoite types. begin to differentiate until about 11-12 days. The oocyst is unique in that it is pedunculated. The peduncle, first seen when the oocyst attains a LITERATURE CITED diameter of about 30 micra (7-8 days), develops Dhanapala, S.B. 1962. The occurrence of to a length of about 25 micra and about the same Plasmodium juxtanucleare Versiani and Gomes, width. The oocyst then matures, attaining a dia- 1941 in domestic fowls in Ceylon. Riv. Malariol., meter of 90-130 micra at differentiation; differ- 41:3-10.

Dr. J. W. Field, Mr. Yap Loy Fong and Dr. A. A. Sandosham A RAPID METHOD OF STAINING THIN BLOOD FILMS FOR MALARIA DIAGNOSIS

MATERIAL AND METHODS (6) Give the slide a final rinse in water, then The requirements are (a) 0.2 per cent eosin allow it to drain and dry before examina- (yellow, water soluble) in methanol (Analar), (b) tion. Field's Stain 'A', and (c) phosphate buffer solu- tion, pH 6.6. RESULTS (I) Put the thin film level on a staining rack, The thin films stained by this method demon- then pour 6 drops of methanolic eosin strated compared favourably with those by the onto the film from a drop bottle. conventional Giemsa method which requires a (2) Immediately add 12 drops of Stain 'A'. longer staining time of half an hour or so. (3) Mix the stain by rocking the slide gently Schüffner's dots are easily demonstrable in the and stain for 1-5 minutes. vivax-like Plasmodium cynomolgi bastianellii (4) Do not pour away the stain but wash by and Maurer's spots discernible in the falcipa- immersing the entire slide in clean running rum-like P. coatneyi infected erythrocytes. The water or flush off with a jet of water from only minor drawbacks are that the films some- a wash bottle. times do not stain uniformly and occasionally (5) Differentiate the stained film in phosphate they are bestrewn with stain debris despite the solution, pH 6.6 for 5 seconds. precaution taken in washing.

Mr. S. Sivanandam, Mr. Yap Loy Fong and Dr. A. A. Sandosham A RAPID METHOD OF STAINING MICROFILARIAE IN THICK BLOOD FILMS

METHOD Stand the slide on end to drain and dry. Then When the thick blood film is dry after 30 fix the dehaemoglobinized film with methanol minutes or longer (or even overnight), dehaemo- for a few seconds and dip the slide in water to globinize it in water for two or three minutes. wash away the methanol before staining. 174 SINGAPORE MEDICAL JOURNAL

(i) Immerse the film in Field's Stain 'A' for clei and tapering tail. Usually it shows a charac- 1 second. teristic unstained margin around the parasites (ii) Wash in water for a few seconds. and occasionally a faint pinkish sheath at the

(iii) Immerse in Field's Stain 'B' for 1 second. posterior extremity. (iv) Repeat wash in water. The routine thick -film rapid staining method (v) Counterstain in Stain 'A' for 1 second. for malaria diagnosis as described by Field (vi) Final wash in water until excess stain (1941) usually fails to stain the microfilariae. disappears. This simple rapid method would provide the (vii) Place the slide on end to drain and dry. medical laboratory workers, especially in estates and during field surveys, with an easily available RESULT and rapid means of establishing diagnosis of filariasis The thick films stained by this method appear other than depending on Giemsa or to produce a better colour result and uniformity haematoxylin stains which require overnight than by the Giemsa method. The microfilariae drying and longer staining time. of Brugia malayi and B. pahangi, stain a bril- The only noticeable drawbacks are the occa- liant purple with well preserved outlines and sional contamination of the films with cellular well-defined body and terminal nuclei; the or stain debris; films which are too thick will not sheaths show a brilliant stippled pink which dehaemoglobinize and stain well. stand out prominently against -a background of deep purple leucocytes and pale pinkish lysed erythrocytes. REFERENCE Field, J.W. (1941). Further note on a method of stain- The microfilariae of Wuchereria bancrofti ing malarial parasites in thick blood films. Trans. stain purple with well-defined discrete body nu- R. Soc. trop. Med. Hyg., 35, 35-42.

Mr. G. L. Coombs and Dr. A. A. Sandosham A MODIFICATION OF FIELD'S RAPID STAIN WHICH MAY BE USED IN STAINING THICK AND THIN BLOOD FILMS MADE SEPARATELY OR ON THE SAME SLIDE

MODIFICATIONS Potassium Dihydrogen Phosphate The modification of Field's Stains recommen- KH2PO, 6.25 gm. ded in this technique are that the stains are made Sodium Chloride 4.25 gm. solution and in 0.85 per cent sodium chloride Distilled water 500 ml. the quantity of Azure I in Field's Stain 'A' is increased from 0.5 to 0.6 gm. TECHNIQUE OF STAINING THIN FILMS SOLUTION "A" Before staining the thin films have to be fixed by methanol and in using combined thin Methylene blue (Medicinal) - 0.8 gm. and thick films precaution has to be taken to prevent I 0.6 gm. Azure fixation of the thick film by contamination. Du- Disodium Hydrogen Phosphate ring the standing and drying processes of the Na2HPO, 5.0 gm. combined thin and thick films, the slide should Potassium Dihydrogen Phosphate he held with the thick film below. KH2PO, 6.25 gm. Sodium Chloride 4.25 gm. METHOD OF STAINING Distilled water 500 ml. (1) Immerse the slide as soon as dry in solu- tion "A" for 1 second. SOLUTION "B" (2) Rinse in running tap water until excess Eosin 1.0 gm. stain is removed. Disodium Hydrogen Phosphate (3) Immerse the film in Stain "B" for 2 to 3 Na2HPO, 5.0 gm. seconds. DECEMBER, 1963 175

(4) Rinse as in (2). dish pink and the females a pale blue. Blood (5) Immerse the film in Stain "A" for 10 to films of P. cynomolgi bastianellii showed mark- 15 seconds. ed Schuffners stippling even with early tropho- (6) Rinse as in (2) and place the slide to dry zoites but in blood films of P. coatneyi Maurer's on a rack. dots were only occasionally seen. In the thick blood films the leucocytes stained STAINING RESULTS a deep purplish blue, the chromatin a deep red The vivax-like Plasmodium cynomolgi bas- and the cytoplasm a purplish blue on a pink tianellii and the falciparum-like P. coatneyi were background. Schüffner's stippling was also seen. used to test the stain and stained slides of these No distortion to the parasites or the leucocytes in thin and thick films were demonstrated. was observed. In the thin blood films the erythrocytes stain- ed a pinkish grey and did not appear overstained REFERENCES and the nuclei of the leucocytes appeared a deep Field, J.W. 1941. Further note on a method of purple. Trophozoites and schizonts staining malaria parasites in thick blood films. appeared Trans. R. Soc. trop. Med. Hyg., 35: 35-42. well stained with the chromatin a deep red and 2. Singh, J. and Bhattacharji, J.M. 1944. Rapid stain- the cytoplasm a deep blue. The gametocytes ap- ing of malaria parasites with water soluble peared well stained with the males being a red- stain. Indian Medical Gaz., 79: 102-104.

By Prof. R. S. Desowitz and Dr. I. Polunin THE INCIDENCE OF INTESTINAL PARASITES IN LAOS A PRELIMINARY REPORT

Very little is known regarding the incidence No. examined 90 and nature of intestinal parasitism in Laos. As No. positive for parasites 85 (94.5%) part of a general medical survey of the Laotian population one of us (I.P.) collected 90 stool Ascaris 72 (80.0%) samples from Pak Lay, a village about on the Hookworm 38 (42.2%) Mekong River. The stools were preserved in MIF fluid and sent by air to Singapore where Trichuris 6 ( 6.6%) they were examined. The present report gives Strongyloides 7 ( 7.7%) the incidence of various parasites found by a single, direct -smear examination of the samples. Taenia 6 ( 6.6%) The results indicate the high incidence of para- Clonorchis/Opistorchis - 24 (26.6%) sitism in the area. The stools are now being examined by concentration (MIF-C). Protozoan cysts 15 (16.6%)

By Dr. F. L. Dunn and Dr. J. M. Bolton THE MIF DIRECT SMEAR (DS) METHOD IN THE STUDY OF INTESTINAL PARASITISM IN MALAYAN ABORIGINES

In August 1962 an investigation of intestinal the 100 bed Aborigines Hospital at Gombak parasitism in Malayan Aborigines was initiated near Kuala Lumpur. Treatment results are as- by the Institute for Medical Research and the sessed by followup faecal examinations. 2) A medical staff of the Department of Aborigines. survey of 200 consecutive admissions, for all The work has proceeded at three levels: 1) Con- causes, to the Gombak hospital-to provide pre- tinuing routine parasitologic diagnosis for all liminary information about the species of intes- admissions (with signs, symptoms, or laboratory tinal parasites occurring in Aborigines, and to findings suggestive of intestinal parasitism) to establish crude prevalence baselines. 3) A series 176 SINGAPORE MEDICAL JOURNAL of field surveys of selected Aboriginal popula- part by the Office of the Surgeon General, Dept. tions in a variety of habitats. Five such surveys of the Army.) (covering 436 persons) have been completed to date. Each survey has been large enough, cover- Appended is a list of parasites now known ing all age groups and both sexes, to serve as a to occur in Malayan Aborigines. Those marked statistically valid sample of the surveyed popu- (*) have not previously been recorded in these lation. Three more surveys are planned. When people. Balantidium coli, and several other pro- these are completed, and all specimens have tozoa, have not previously been recorded from been studied, approximately 900 individuals man in the Federation of Malaya (although they (2% of the total population of c. 45,000) will have been recognized in Singapore). have been examined at least once for intestinal Helminths - hookworm, Ascaris lumbricoi- parasites. The surveys will provide the following des, Trichuris trichiura, Ente- information (correlated with habitat and mode robius vermicularis, Taenia sp., of life): 1) the kinds of parasites present, 2) pre- Hymenolepis nana*, Tricho- valence rates for these parasites, and 3) data on strongylus sp.* Strongyloides intensity of infection for Ascaris, Trichuris, and stercoralis, unknown dicrocoe- hookworm (based on egg counts). The results of lid trematode-species. these surveys will provide guidelines for future 1. *, unknown trematode-spe- mass treatment campaigns the in field. As a final cies 2. *, Dipylidium caninum phase of the investigation, pilot mass treatment has also been reported in Abo- projects will be carried out in several of the sur- rigines. veyed communities (with periodic resurvey to Protozoa Entamoeba histolytica, large determine rates of reinfection and intensity of - reinfection). race; Entamoeba histolytica, small race = E. hartmanni*, The basic procedure in all of these investiga- Entamoeba coli*, Endolimax tions is the MIF-DS method of examination of nana*, lodamoeba bütschlii*, faecal specimens. The demonstration illustrates Dientamoeba Jragilis*, Giardia the steps involved in preparing MIF sets, and the lamblia*, Chilomastix mesnili*, procedures involved in handling and examining Trichomonas hominis*, Isospo- the specimens. The advantages and disadvan- ra sp. *, Balantidium coli *. tages of the procedure are outlined. It is empha- sized that the MIF-DS method is invaluable REFERENCES not only for field survey use, but also for Sápero, J.J. and Lawless, D.K. 1953. Amer. J. Trop. routine Med. Hyg. 2: 613-619. in small hospitals and other medical units in ru- Blagg, W. et al. 1955. Amer. J. Trop. Med. Hyg. 4: ral areas where laboratory facilities may be 23-28. limited. The method is simple and, with proper Kuntz, R.E. 1960. Amer. J. Trop. Med. Hyg. 9: 168- precautions, at least as reliable as most of the 172. well-known (and often more complex) proce- Kuntz, R.E. and Wells, W.H. 1962. Amer. J. Trop. Med. Hyg. II: 773-780. dures commonly in use. (The studies outlined above Sandosham, A.A. 1953. Malaysian Parasites XIV. are being supported in part by US Public Stud. Inst. Med. Res. Malaya 26: Health 210-211. Service ICMRT Grant AI -04189-02; in Polunin, I. 1953. Med. J. Malaya 8: 55-174.

Mr. C. P. Ramachandran, Dr. F. L. Dunn, Professor A. A. Sandosham and Mr. S. Sivanandam A DIROFILARIA FROM THE MUSANG

In the course of the filariasis investigations periodic Brugia rnalayi and Brugia pahangi undertaken by the Institute for Medical Re- (Laing, Edeson, and Wharton, 1960). In addition search in recent years, various wild and domestic Musangs have often been found infected with animals have been examined in the East Pahang another filarial worm, usually recovered from area of Malaya for infections with Brugia spp. the sub -cutaneous tissues. From very brief mor- The civet cat, Paradoxurus hermaphroditus phological studies (of microfilariae only) these (Carnivora), commonly known as the Musang, worms have been regarded (Wharton, 1962) as has been found infected in nature with sub - Dirofilaria repens. No detailed studies of the DECEMBER, 1963 177 adult worm from Musangs have been under- host to host. Size differences and proportions in taken in the past. In this demonstration some structures such as the spicules, however, are less preliminary results of such a study are presented. subject to variation. The left spicule in the genus Dirofilaria is divided into a proximal and a dis- These worms conform in general characters tal part. In D. repens the distal portion is about to the Genus Dirofilaria (Railliet and Henry, 1.5 times the length of the proximal, while in 1911) which contains a number of - species paras the Musang Dirofilaria the distal portion -is less tic in primates, carnivores, and rodents. Of these than, or approximately equal in length to the the best known are Dirofilaria immitis (Leidy, proximal portion. The whip -lash distal of the 1856), the heart worm of dogs, and Dirofilaria left spicule, characteristic of D. repens, is not repens (Railliet and 1911) Henry, from cats and present in the musang worms. These differences dogs. Webber (1955), and Chabaud - and Rousse in form and structure, in conjunction with the lot (1956) have reviewed the Dirofilaria of pri- smaller size of the adult worms, suggest to us mates. Price (1959) has described Dirofilaria that the Musang Dirofilaria is probably an un - magnilarvatum, from a Malayan Macaca irus. described species, distinct from but closely re- Rohde (1962) has the studied helminths of cats lated to D. repens. The microfilariae resemble and dogs in Malaya has and reported D. repens- closely the published description of those of D. like nematodes from a domestic cat. Dr. Rohde repens and D. immitis. Further work on this of the Department of Zoology, University of worm is continuing. Malaya has kindly permitted us to examine his specimens which in most respects conform to REFERENCES the classical description of D. repens. Both the Chabaud, A.G., and Rousselot, R. (1956). Ann. males and females, however, are slightly shorter Parasit. 30, 53. than in earlier descriptions. 2. Laing, A.B.G., Edeson, J.F.B., and Wharton, R.H. (1960) Ann. trop. Med. Parasit. 54, 92. None of the descriptions of Dirofilaria pre- 3. Price, D.L. (1959). J. Parasit. 45, 499. viously known correspond closely to the present 4. Rohde, forms recovered from the civet cat. Three com- K. (1962). Z. of Parasitenkunde. 22, 237. 5. Sandosham, A.A. (1951). J. 25, 19. plete males (and one fragment) and 14 complete Helmintlt. females have been studied. The measurements, 6. Taylor, A.E.R. (1960). J. Helminth. 34, 13. which are fairly consistent, indicate that these 7. Webber, W.A.F. (1955). Ann. trop. Med. Parasit. 49, 123. worms are much smaller in size than D. immitis 8. Wharton, R.H. Bull and D. repens. It may be suggested that the (1962). Inst. Med. Res. size Federation of Malaya. No. II of 1962. p. 82, of the worm is only relative, and may vary from and 91.

Dr. K. Rohde (Introduced by Professor A. A. Sandosham) TREMATODES OF BATS IN MALAYA (BAT AS HOST OF HUMAN PARASITE?)

42 bats, comprising 8 species, were examined cularis in a bat. This indicates that the genus for helminths. The following trematodes were Odeningotrema Rohde, 1962, subfam. Odenin- encountered: Cephalotrema sp. (?) (Prosthogo- gotrematinae Rohde, 1962, has a very low de- nimidae), Postorchigenes duboisi n.sp., Leci- gree of host -specificity. It has been found in thodendrium linstowi Dollfus, 1931, Prostho- Primates (Tupaiidae, Lorisidae), Chiroptera dendrium longiforme Bhalerao, 1926, P. parvou- (Molossidae), and Insectivora (Erinaceidae). terus Bhalerao, 1926, P. swansoni Macy 1936, (Rohde, in press). Odeningotrema bivesicularis Rohde, 1962, and In the meantime, a large number of trema- small unidentified Lecithodendriidae. All species todes from 5 specimens of a 9th species of bats, except the first belong to the family Lecithoden- Taphozous melanopogon, were examined. Four driidae. Postorchigenes duboisi n.sp. is the first species of Lecithodendriidae, belonging to the member of this genus from mammals. Of special genus Prosthodendrium (2 of them to the sub- interest is the finding of Odeningotrema bivesi- genus Paralecithodendrium) were found. Most 17s SINGAPORE MEDICAL JOURNAL common is a form closely resembling P. (Para- Taphozous must be considered a reservoir host lecithodendrium) rnolenkampi (Lie-Kian-Joe, of this (accidental?) human parasite. 1951). It is very probably identical with this species, which was described as a species of the REFERENCES genus Paralecithodendrium from man in Indo- Dubois, G.: Rev. Suisse de Zool. 69, 385-407 (1962). nesia and later (comp. Dubois, 1962) put into Lie -Klan -Joe: Doc. Neerl et lndon. de Morb. Trop. the genus Prosthodendrium, subgenus Paraleci- 3, 105-116. (1951). thodendrium (see exhibit and figure Il). Thus, Rohde, K.: Z Parasitenkunde (in press).

Dr. Hong -Fang Lee TWO NEW TREMATODES OF FERAL RATS IN MALAYA AND NORTH BORNEO

Five species of trematodes were recovered gonimus, but the structure and position of the from feral rats trapped in Malaya and North other organs exclude them from this genus. They Borneo. Skrjabinus muris Stscherbakova, 1942 resemble Paragonimus in spination, position of and Athesmia foxi Goldberger and Crane, 1911 ovary, testes, uterus and distribution of vitella - (both Dicrocoeliidae), were found for the first ria, but they differ in that they have smaller eggs time in Malaya and the rats (R. sabanus, R. ar- and Y-shaped excretory vesicle. The generic gentiventer and R. rajah surifer) represented new name Beavertrema is proposed for these species hosts; Echinostoma malayanurn, a common in- in honor of Professor Paul C. Beaver of Tulane testinal fluke of Malayan rats, was found in R. University. argentiventer. Two new species of a new genus, beaveri was recovered from both tentatively assigned to the family Troglotrema- Beavertrema trapped in R. liver and intestines of R. sabanus tidae, were recovered from R. sabanus and was found infraluteus. Malaya while B. microacetabulum only in the liver of R. infraluteus from North The two new trematodes do not belong to any Borneo. B. microacetabulum is distinguished of the described genera. The position of the geni- from B. beaveri by a smaller acetabulum which tal pore is similar to that in the genus Prostho- is closer to the oral sucker.

Dr. P. F. Basch (Introduced by Prof. A. A. Sandosham) AN AVIAN SCHISTOSOME FROM NEGRI SEMBILAN PADI FIELDS

Collections of freshwater snails, Lymnaea sp., proved to be suitable hosts, as shown by mira- from padi fields in the vicinity of Kuala Pilah, cidia hatched from their faeces. Of five mature Negri Sembilan, have revealed a great many tre- ducks purchased in Kuala Pilah, one had a. light matodes present in the fields. There are several infection, and one a very slight case, as demon- species of echinostomes, at least one small stylet strated by hatching of miracidia. One experi- cercada, at least two strigeids, and an eyed schi- mental duckling has been sacrificed, and in spite stosome cercada of the "cercada ocellata" of an intensive search no adult worms were lo- group. This latter cercaria is capable of causing cated; more animals are now being given very a dermatitis in man, as was pointed out by Dr. heavy exposures in hopes on obtaining the adult Sandosham in 1953 (Malaysian Parasites XII). worms and eggs. The adult seems to be a species Large numbers of snails have been collected and of Trichobilharzia. One observation of interest checked, and about 12 naturally infected snails in the laboratory has been the finding in small are regularly shedding these cercariae in the la- free-living rhabdocoel flatworms of numbers of boratory. Experimental exposures have been cercariae, alive and moving. Whether the worms. made to ducks, doves, sparrows, munias, finches, eat the cercariae, or whether the latter penetrate and mice, but thus far only ducklings have actively is still not known. Attempts have been DECEMBER, 1963 179 made to establish the life cycle in the laboratory Any specimens or information about avian or by exposing clean lab -raised snails to miracidia mammalian schistosomes in Malaysia would be from naturally and experimentally infected gratefully received by the writer at the address ducks. above.

Mr. Lim Boo Liat, Mr. Ow -Yang Chee Kong and Prof. Lie Kian Joe (Introduced by Prof. A. A. Sandosham) FURTHER RESULTS OF STUDIES ON ANGIOSTRONGYLUS CANTONENSIS CHEN, 1935 IN MALAYA

Angiostrongylus cantonensis has been found ed under experimental conditions in the labora- in rats in various habitats around Kuala Lum- tory. They may be infected with first stage larvae pur: scrub, lallang, rice field and oil palm es- which will develop into third stage larvae in tates. Land snails and slugs collected from these these snails. They may also be infected with areas were dissected and the results were shown third stage larvae which remain as such in the in tables 1, 2, 3 and 4. Third stage larvae were snail without further development. Table 6 found in Microparmarion malayanum, Parma- showed the results of experiments with first stage rion sp. and Pseudoplecto bijuga. larvae and table 7 with third stage larvae. It is found that the rats do swallow the slugs Fresh water snails do eat dead slugs and land and land snails in the laboratory as well as in snails. Infection of fresh water snails in nature the field. Moreover, rat faeces seem to form part may occur from eating dead slugs or land snails the of food taken by land snails and slugs. The or perhaps from infection with first stage larvae. relationship between rats and the intermediate Pila scutata and Bellamya snails are eaten by hosts is therefore direct and mutual. man. In certain countries they are eaten raw. In addition, third stage larvae were found in They are therefore a possible source for human fresh water snails collected from padi fields. Re- infection with Angiostrongylus cantonensis. At- sults of dissections of these snails were shown tempts to infect fresh -water shrimps with larvae in table 5. These fresh water snails can be infect- of Angiostrongylus have failed.

Prof. Lie Kian Joe (Introduced by Prof. A. A. Sandosham) THE LIFE HISTORY OF A HYPODERAEUM sp. (TREMATODA: ECHINOSTOMATIDAE)

The complete life cycle of a Hypoderaeum culus, Indoplanorbis exustus) and tadpoles species a parasite of ducks and geese, has been (Rhacophorus leucomystax). Drawings were worked out. The freshwater snail Lymnaea ru- shown of the sporocyst, mother redia, redia con- biginosa serves as the first intermediate host. taining rediae and cercariae, cercada, and me- The sporocyst is found in the heart muscle. tacercariariae. A stained adult worm was also There are at least 3 redial generations. The shown. The parasite is described as a new spe- second intermediate hosts are different fresh- cies in Tropical and Geographical Medicine (in water snails (L. rubiginosa, Gyraulus convexius- press). 180 SINGAPORE MEDICAL JOURNAL

Mr. Cheong Wéng Hooi and Mr. C. P. Ramachandran (Introduced by Prof. A. A. Sandosham) (MERMETHID?) FROM TWO MALAYAN MOSQUITOES

During the course of our entomological in- larval forms. Agamomermis sp. have been col- vestigations on Malaria and Filariasis, various lected from the abdominal cavity of larvae, pu- filarial and nematode worms of unknown gen- pae and adults of Culex nemorales Stiles (1903). eric or specific identity have been recovered Smith (1904) have reported the occurrence of from mosquitoes. As far as we know, no pre- Agamomermis culicis from Aedes sollicitans in vious reports have been made on these findings. New Jersey. Many species of mermithid nema- The present demonstrations show two of such todes have been reported from mosquitoes but unknown nematodes recovered from: little is known of their biology. Lyengar (1930) (a) Anopheles letifer: Wild caught at Bukit has also noted the occurrence of certain large Kelubi in Selangor on 22/ 11 /61. The species of Mermis from adult mosquitoes. A unknown nematode was found in the mermithid nematode was described in Aedes abdominal haemocoele. communis in Canada, Jenkins and West. (1954). See slide 1. These appear to be the first records of rather Length : 17.35 m.m. similar infections from Malaya. Breadth : 0.12 m.m. (b) Aedes albopictus: Wild caught at Ulu Gombak in Selangor on 8.6.63. The un- REFERENCES known nematode was recovered from Stiles. C.W. (1903). U.S.P.H. Mar. Hosp. Serv., the abdominal haemocoele. Hyg., Lab. Bull. 13: 15-17. See slide 2. 2. Smith, J.B. (1904). Report of the New Jersey State Agricultural experiment station. Trenton, N.J. Length: 11.1 m.m. 3. Iyengar, M.O.T. (1930). Trans. F.E.A.T.M. 7th Breadth : 0.145 m.m. Congr. Calcutta, 3, 128-42. A number of workers have reported nematode 4. Jenkins, D.W. and West, A.S. (1954). Mosquito parasitism of adult mosquitoes as well as in the News, 14, 138.

Prof. Lie Kian Joe (Introduced by Prof. A. A. Sandosham) THE LIFE HISTORY OF ECHINOSTOMA LINDOENSE SANDGROUND AND BONNE, 1940 (TREMATODA; ECHINOSTOMATIDAE)

Echinostoma lindoense is a human parasite water snail Gyraulus convexiusculus serves as found among the inhabitants around Lake Lin - first intermediate host. The sporocyst develops doe in Central Celebes, Indonesia (Sandground in the heart muscle. There are 2 redial genera- and Bonne, Am. J. Trop. Med., 1940, 20, 511). tions. The second intermediate hosts are fresh- The life cycle of the parasite in that region was water snails (Lymnaea rubiginosa, Gyraulus partly worked out. The first intermediate host convexiusculus, Indoplanorbis exustus) and tad- is Gyraulus sarasinorum and the second inter- poles (Rhacophorus leucomystax). The parasite mediate hosts are various freshwater snails and is maintained in nature in Malaya by domestic a mussel Corbicula lindoensis Boll. Man gets in- ducks and probably also geese. White mice are fected by eating the infected mussels. suitable experimental hosts. The parasite is not reported in man yet in this country. Drawings E. lindoense is now found in Malaya, the first of a sporocyst, mother redia, daughter redia, cer- record of E. lindoense, outside the Lindoe area. ceria, and metacercaria were shown. A stained The complete life cycle is worked out. The fresh- adult worm was also demonstrated. DECEMBER, 1963 181

Mr. Ow Yang Chee Kong (Introduced by Prof. A. A. Sandosham) A SPECIES OF HAPLORCHIS FROM THE WATER MONITER

Since the establishment of Procerovum by A stained specimen of this worm was exhibit- Orji and Nishio (1924) and Haplorchoides by ed. Chen (1949), many species of heterophyid tre- REFERENCES matodes that had been referred to the genus Africa, C.M. (1938). Description of three trematodes Haplorchis Looss, were transferred of the genus Haplorchis (Heterophyidae) with notes to these two on two other Philippine members of this genus. genera. On the basis of this reclassification, only Philipp. J. Sci. 66: 299-306. 6 species that Morozov (1952) listed as distinct, Chen, H.T. (1936). A study of the Haplorchinae still remain in the original genus. (Looss 1899) Poche 1926 (Trematoda: Hetero- phyidae). Parasit. 28: 40-55. A species of Haplorchis Looss, s.s. which does Chen, H.T. (1949). Systematic consideration of some heterophyid not appear fit Trematodes in the subfamilies Haplor- to into any of the existing species chinae and Stellantchasminae. Ann. Trop. Med. has now been found in Varanus salvator (Lau- Parasit. 43: 304-312. renti) from Malacca. Incidentally, this is also Orji, K. and Nishio, T. (1924). Ueber die Distomen, the first record from a reptilian host, those re- welche den Darmcanal bewohen. Mitt. Med. Ges. Chiba. 2: 351 (vide Chen, 1949). ported previously from being warm blooded Morozov, F.N. (1952). Trematodes of the superfamily animals only; including at least 4 species from Heterophyoidea Faust, 1929. In K. I. Skrjabin: man-namely, H. Pumilio (Looss), H. taichui Trematodes of animals and man. Vol. 6: pp. 153- (Nishigori), H. vanissimus Africa, and H. yoko- 351. Yamaguti, S. (1958). The gawai (vide digenetic trematodes of ver- (Katsuta) Yamaguti, 1961). tebrates. In Systema Helminthum, vol. 1.

Dr. K. Rohde (Introduced by Professor A. A. Sandosham) STUDIES ON THE GENITAL SYSTEM OF MONOGENETIC TREMATODES

Recently the author described the first Ma- main part of the ootype. In addition, numerous layan monogenetic trematode, belonging to the small nuclei of the ordinary parenchymatous genus Polystomoides Ward, 1917, from the uri- type are distributed mainly in the connective tis- nary bladder of the tortoise Cyclemys amboin- sue, situated between the serous glands, and ensis (Daud.) (Rohde, 1963; see exhibit 1 and around the anterior and posterior entrances into figure 1). In the meantime, 3 further species of the ootype. Similar nuclei are in the epithelium the same genus have been found. Descriptions of the ootype. As a working hypothesis it is as- of these species and a revision of the whole genus sumed that these nuclei migrate into the ootype, are being prepared. forming the (temporary?, see below) epithelium, which is eventually shed into the lumen of the The presence of 4 species of the same genus ootype (figures 4, 5, 6). According to Kohl- in Malaya, occurring in sufficient - numbers, ap- mann (1961) the same types of cells can be found pears to provide a good opportunity for compa- in the closely related form Polystomminteger- rative histological and cytological studies of rimuln Fröhlich: mucous glands at the entrance Monogenea. Histology and cytology of this of the ductus communis into the ootype, and group are very poorly known (Bychowsky 1957). serous glands opening into the ootype, which is surrounded by an In the following, some preliminary results of epithelium. comparative histological studies on the ootype, In the smaller species of Polystomoides mu- and the accessory male glands of Polystomoides cous gland cells around the entrance of the duc - spp. are communicated. tus communis into the ootype and numerous small, undifferentiated nuclei around the ootype (A) Ootype. In the largest species (P. malayí), are also present. But though serous secretion there are mucous gland cells, which open into can always be found in the lumen of the ootype, the ductus communis just in front of its entrance serous gland cells can usually not or only with into the ootype (figures 2, 3), and large serous difficulty be distinguished. Even if they are dis- glands with huge nuclei, which open into the tinguishable, they are much smaller and much 182 SINGAPORE. MEDICAL JOURNAL more similar to the ordinary parenchymatous other; not yet verified!) resembling the serous type of cells than the serous glands in P. malayi. glands of P. malayi! The 2 smallest species have A continuous epithelium around the ootype no morphologically distinct accessory male of at least the 2 smallest species is absent, though glands at all, though in these cases there is al- there are always a few nuclei inside the lumen, ways a cluster of small nuclei, similar to the pa- which have probably immigrated from the sur- renchymatous ones, near the genital bulb. Poly- rounding tissue (see above, figure 8). stomum integerrimum, according to Kohlmann, 1961, has only 1 type of accessory male glands. The structure of the ootype in the 3 small species of Polystomoides appears to correspond Hence, the presence of certain types of ootype in many points to the structure of the ootype in glands and accessory male glands in Polystoma immature P. malayi (figure 7). and Polystomoides is not a generic character, but varies greatly, depending on the size of the (B) Accessory male glands. The same varia- parasite. bility among the 4 species of Polystomoides can be found in the accessory male glands. P. ma- REFERENCES layi, the largest species, has two types of acces- Dychowsky, B.E.: Monogenetic Trematodes. 1957. sory glands, i.e. mucous and serous glands. The Kohlmann, F.W.: Z. Parasitenkunde, 20, 495-524 second largest species has only 1 type (or per- (1961). haps 2 types which are very similar to each Rohde, K.: Z. Parasitenkunde, 22, 278-282 (1963).

Mrs. V. Thomas (Introduced by Prof. A. A. Sandosham) THE VARIATION IN DDT -RESISTANCE IN LARVAE OF DIRECTLY AND INDIRECTLY SELECTED COLONIES OF CULEX PIPIENS FATIGANS WIED

Two methods of selection were adopted. One subsequent generations had not been subjected was by direct selection when larvae were hatch- to selection by exposure to DDT. ed out in bulk from many egg rafts obtained In the first colony where the larvae from Klang and submitted to a discrimination were iso- lated and maintained under DDT -selection pres- concentration of 5 p.p.m. of DDT. The adults sure in successive generations, resistance that emerged from the survivors were maintain- to DDT rose to a high level. In the colonies of the ed as a resistant colony and their larvae in suc- second group which had been selected solely cessive generations were subjected to the same because of high resistance of their sibs to DDT, concentration over a number of generations. the resistance was maintained only through the The other method of selection was an indirect first 2 to 4 generations and subsequently lost one. Larvae hatched out from individual egg completely by the 5th generation (see graph). rafts collected from Klang were reared separate- The LC value in the directly selected colony was 14.5 ly and about 50 larvae from each raft were sub- p.p.m. for the sixth generation whereas it had fallen in the indirectly selected colonies jected to 5 p.p.m. of DDT to determine the mor- to 0.33 and 0.28 p.p.m. by the fifth generation. tality rate. The remaining unexposed larvae of individual batches that showed the lowest mor- It was noted that the building up of resistance tality rate to this concentration were maintained even under selection pressure was fluctuating as separate colonies for several generations. and irregular and that there was rapid reversion Three colonies were established and changes in to susceptibility in untreated larvae. These ex- mortality rate in successive generations were perimental findings suggest that DDT -resistance observed on about 50 larvae of 10 individual in larvae of C. p. fatigans is due to cytoplasmic rafts from these colonies. In this process of in- modification and were confirmed in reciprocal direct selection the larvae which gave rise to the and back -cross experiments.