Cyclically transmitted infections of Trypanosoma brucei, T. rhodesiense and T. Gambiense in chimpanzees

781 TRANSACTIONSOF THE ROYAL SOCIETYOF TROPICAL MEDICINE AND HYGIENE. Vol. 61. No. 6. 1967.

CYCLICALLY TRANSMITTED INFECTIONS OF TRYPANOSOMA BRUCEI, T. RHODESIENSE AND T. GAMBIENSE IN CHIMPANZEES D. G. GODFREY* AND R. KILLICK-KENDRICK**

Nigerian Institute for Trypanosomiasis Research, Vom, Northern Nigeria Trypanosoma rhodesiense and T. gambiense closely resemble T. brucd in both morphology and behaviour. The only outstanding difference is the inability of T. brucei to infect man, although, otherwise, all three species can infect a sjmilar wide range of mammals. Other differences are not so well defined and vary according to the strains studied. Generally, T. rhodesieme causes a subacute infection in man in contrast to the typical chronic infection of T. gambiense; this difference is also seen with recently isolated strains in laboratory animals, T: brucd behaving like T. rhodesiense. Many species of mammals have been used as hosts in experimental work with these members of the subgenus Trypanozoon (HoARE, 1964), but, despite the importance of man's susceptibility as a method of differentiation, only a few observations have been made on experimental infections in the anthropoid apes. THOMASand LI~rOlq (1909) described an infection of T. gambiense in a chimpanzee but it was not until recently that BAKER (1964) found the chimpanzee to be unlike man in being susceptible to both T. rhodesiense and T. brucei. These workers, however, infected their apes with parasitized blood, and, as no infections transmitted by tsetse had been described up to 1962, when some chimpanzees became available at this Institute, we decided to compare cyclically transmitted infections of T. rhodesiense, T. gambiense and a proved T. bruce/. Since then, however, a fly-transmitted infection of T. gambiense in a chimpanzee has been briefly described by LvcassE (1964).

Chimpanzees

Chimpanzee 1. The first chimpanzee (Pan satyrus) was a male presented to the Institute, the donor having kept the animal for about one year. The chimpanzee was not infected until nearly 2 months after arrival; during this period it was very active, healthy and gained a ldlogramme in weight. The animal weighed 21.0 kg. when it was infected with T. brucd. Chimpanzee 2. A male chimpanzee was purchased and kept for 10 months before being used. It was very active and healthy during the pre-experimental period, although *Now at the Lister Institute of Preventive Medicine, Chelsea Bridge Road, London, S.W.1. **Now at the Department of Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, W.C.1. We thank Mr. C. J. Roberts for conducting an autopsy on one chimpanzee. We also wish to acknowledge" the technical assistance of Mallam H. M. Yesufu And Mr. S. Jikeme, and the encouragement of our former Director, Dr. K; C. Willett, and the present Director, Mr. T. M. Leach. We are particularly indebted to our wives for their assistance in befriending the chimpanzees, thereby ensuring the animals' co-operation in routine procedures.

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CYCLICALLY TRANSMITTED TRYPANOSOME INFECTIONS I N CHIMPANZEES

microfilariae were occasionally seen in the blood. During the last 2 months before use, the animal increased by 1.8 kg. in weight to 18.1 kg., when it was infected with T. rhodesiense. Chimpanzee 3. A small male chimpanzee was purchased which was very active and healthy during the pre-experimental period of nearly a year. Microfllariae and crescents of Plasmodium rdchenowi were sometimes seen in the blood, and a single dose of 200 mg. amodiaquine base was given orally 12 days before the infective tsetse fly fed. During the final 2 months of the pre-experimental period the animal gained 1.2 kg., and weighed 10.7 kg. at the time of infection with T. gambiense.

Tsetse flies Pupae of Glossina palpalis were sent to Vom from the Institute's Entomology Section at Kaduna. They were kept at 28°C. and, whenever possible, were offered the first infected feed on the first day of emergence.

Trypanosomes T. brucei. Isolation 8/18 of T. brucei was inoculated from a naturally infected pig into a rat at Nsukka in Eastern Nigeria on 12 September 1962 (KILLICK-KENDRICKand GODFm~Y, 1963). 7 days later, polymorphic trypanos0mes were seen in the rat's blood and high parasitaemias eventually developed; postero-nuclear forms were seen. These observations suggested that the trypanosome was T. brucei, not T. gambiense which usually has a low infectivity to rats and which rarely develops postero-nuclear forms. The trypanosome was unlikely to have been T. rhodesiense as this species does not normally occur in West Africa. However, as the only certain way of differentiating T. brucei from T. rhodesiense and T. gambiense is to demonstrate its inability to infect man, 2 experiments were carried out on 2 persons who had not previously been infected by trypanosomes. On 3 November 1962, a tsetse fly infected with T. brucei 8/18 was offered a feed from a healthy European male, but the fly only probed and did not ingest blood. On the following day, however, the fly fed well on the same person. A marked reaction occurred at the site of the bite, but it was later demonstrated that the volunteer reacted similarly to the bite of an uninfected fly; the reaction was therefore probably not due to invading trypanosomes. During the ensuing month, trypanosomes were never seen in the blood of the volunteer, n o clinical changes occurred,, nor were there any significant variations in body temperature, erythmcyte sedimentation rate, haemoglobin value, and erythrocyte count; the volunteer still remains in good health at the time of writing. 32 days after the infected tsetse fly had fed, 450 ml. of blood were taken from the volunteer into 75 ml. of anticoagulant ('distilled water containing 2.2% w/v sodium citrate, 0.73% w/v citric acid, and 2.45~/o w/v dextrose); 3 ml. of the mixture were inoculated intraperitoneally into each of 2 rats, and similarly 470 ml. into a large dog whose blood had previously failed to infect rats. Neither the rats nor the dog showed any trypanosomes in their blood during the following 4 weeks' examitmtion; the dog had no increase in body temperature and has since remained healthy for over 4 years. Although this result suggested that the trypanosome was not infective to man, the possibility that it was T. rhodesiense could not be excluded on this evidence alone, because it is known that an infection with this trypanosome may not necessarily arise from the bite of just one infected fly (FAIRBAIm~and BtmTT, 1946). These authors considered that any failures were due to an insufficient number of metacyclic trypanosomes being injected. WILLETT(1956) estimated the minimum infective dose for the

D. G. GODFREY AND R. KILLICK-KENDRICK

783

bloodstream forms of T. rhodesiense to be about 20,000 trypanosomes. We therefore inoculated another healthy European male subcutaneously with over 100 times the calcttlated minimum infective dose, viz., 2.8 million trypanosomes in 0.1 ml. rat blood; the donor rat had been infected by the tsetse fly which fed on the first voltmteer. Again trypanosomes were never seen in the blood, nor was there any change in the volunteer's clinical conditions or blood values; he aiso remains in good health at the time of writing. 23 days after the inoculation, 210 ml. of blood were withdrawn into 75 ml. of the anticoagulant solution. 3 ml. of the blood mixture were inoculated intraperitoneally into each of 3 rats, and the remainder intravenously into a horse. Wet blood fiims from the rats and the horse were examined daily for 5 weeks, but trypanosomes were never seen and no increase in the body temperature of the horse was recorded. We concluded that the trypanosome was definitely T. brucei. In order to transmit T. brucei to the chimpanzee, G. palpalis were fed on the rat infected at Nsukka, and also on a sheep subsequently inoculated from the rat. Feeding was continued on the sheep for about 2 weeks, then batches of 10 flies were each fed on a single rat. I f a rat became infected, each fly in the batch was fed on an individual rat, so that eventually the infected flies were identified and isolated. One such fly was used to infect Chimpanzee 1 on 20 November 1962. T. rhodesiense. Isolation 8/8/105A of T. rhodesiense was isolated in Uganda at the East African Trypansomiasis Research Organization and was obtained by us as deepfrozen material through the kindness of Dr. W. E. Ormerod at the London School of Hygiene and Tropical Medicine. During a period of 8 months, 754 tsetse flies were used in unsuccessful attempts to transmit this isolation of T. rhodesiense. A further sample of the isolation was then obtained from Dr. Ormerod, and eventually one infected fly was isolated out of 499 G. palpalis given infected feeds. This fly had fed on an infected rabbit on 4 occasions and was then maintained on a goat for 21 days before being fed on a rat. This rat became infected and metacyclic trypanosomes were also found in the fly by the probe test (BURTT, 1946). On 19 February 1964, the fly fed on Chimpanzee 2, but as no infection was detected by 10 March, the same fly was allowed to feed again; this time transmission was succesful. T. #ambiense. In Benue Province, Northern Nigeria, on 7 November 1962, a blood sample was taken from a patient whose blood and gland juice contained trypanosomes. The blood was inoculated intraperitoneally into a rat. 3 weeks later, one trypanosome was seen in 3 microscope fields ( × 4 0 objective) of a wet blood fdm, and on the following day, one per 60 fields was seen; on all other occasions the rat's blood was microscopically negative. On 7 December 1962, a month after being inoculated, and a week after trypanosomes had been seen, the rat was killed and its blood inoculated into a young patas monkey (Erythrocebus patas). 6 days later trypanosomes appeared in the blood of the monkey and they were seen on many occasions during the following 2 months. G. palpalis were fed on the monkey and eventually an infected fly was found by the probe test. The fly infected Chimpanzee 3 on 29 January 1963.

Techniques Body temperatures.

The daily body temperatures of Chimpanzee 1 was taken orally;

rectally of the other two.

Examination for trypanosomes. A daily wet blood film, taken from the ear, was examined under the ×40 objective of the microscope; 100 microscope fields were examined before the result was classed as negative. A wet film of each specimen of cerebrospinal fluid was also searched before and after centrifugation. Whenever sufficient blood or eerebrospinal fluid was available, rats were inoculated; in the case of Chimpanzee 3, infected with

784

CYCLICALLY TRANSMITTED TRYPANOSOME INFECTIONS IN CHIMPANZEES

7". gambiense, nursling rats were used. The morphology of the trypanosomes found was checked from time to time on Giemsa-stained thin blood films. Blood examinations. The foUowing estimations were made at the times shown in the results: Erythrocyte sedimentation rate. Westergren tubes were used in a constant temperature room (70°F.); readings were made after 30 and 60 minutes. ltaemoglobin was estimated by the Sahli method. Erythrocyte counts were made in an improved Neubauer haemocytometer, with Hayem's fluid as a diluent. Leucocyte counts were made in the haemocytometer after dilution with Toisson's fluid. Gerebrospinal fluid. Whenever lumbar puncture was successful, the white cells in the undiluted fluid were counted in a haemocytometer. Anaesthesia Although some daily routine procedures could be carried out on the chimpanzees after training, venepuncture and lumbar puncture had to be effected under general anaesthesia. On 2 occasions, Chimpanzee 1 was given 500 nag. diethylthiambutene hydrochloride subcutaneously; he became comatose after about 30 minutes and blood was withdrawn from the leg veins. 15 minutes later he reacted to lumbar puncture, and 1 ml. of a local anaesthetic (2.5% w/v ethocaine hydrochloride B.P.) was inoculated near the site of puncture. Quarter of an hour later, 40 rag. of nalorphine hydrobromide B.P. was given subcutaneously and the chimpanzee made a rapid recovery. On one occasion, Chimpanzee 3 was given 40 rag. phencyclidine hydrochioride intramuscularly, which rapidly induced anaesthesia for about 1½ hours. For all other sampling, ~-I grain of morphine sulphate (according to size) was administered subcutaneously, followed about 45 minutes later by pentobarbitone sodium B.P. Chimpanzee 3 was sufficiently trained to receive the barbiturate intravenously at a dosage rate of 9 rag. per kg.; the two others were given the drug intramuscularly at 18 mg. per kg.

Results

Prepatent periods. In the histogram, the day on which the infected fly fed is designated day 0. The infection of T. brucei was first patent on day 6 and T. gambiense on day 9. The tsetse fly that infected Chimpanzee 2 with T. rhodesiense fed twice; as no trypanosomes had appeared in the blood 19 days after the first feed, the fly v~as allowed to feed again; trypanosomes appeared in the blood 8 days later. Reaction at the site of the tsetse fly bite The chimpanzee infected with T. brucei had a slight swelling on day 5 at the site of the bite, which disappeared 2 days later. The chimpanzee with T. rhodesiense had a distinct swelling 2 to 3 ram. in diameter on day 7, which increased in hardness and size by the following day; by day 9 it was distinctly raised with a diameter of 12 ram. but had become softer. The swelling then decreased in size until by day 14 it had disappeared. The sw~dling at the site of the infective bite was more distinct in the T. gambiense infection. On day 4, a red mark, about the size of a pin head, appeared and by day 9 had increased to a diffuse area of 60 ram. diameter with a hard central lump. Day 9 was the first day on which trypanosomes were seen in the peripheral blood. The reaction gradually decreased in size and disappeared by day 13.

Parasitaemia T. brucei multiplied rapidly in the chimpanzee until, on day 12, the equivalent of 4000 trypanosomes were seen in 100 microscope fields of a wet preparation of blood

D. G. GODFREY AND R. KILLICK-KENDRICK

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(see histogram). The numbers then decreased over the next 2 days until, on day 14 when the chimpanzee appeared to be dying, only one trypanosome was seen per 100 fields. The course of infection with T. rhodesiense was a relapsing one with moderately high parasitaemias of up to 1000 trypanosomes per 100 fields. The first peak occurred 5 days after the first day of patency, and subsequent peaks occurred at intervals of 5-10 days until the chimpanzee died on day 42. T ,BRUC~I

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The histogram also shows the chronic course of infection with T. gambimse. The maYimum parasitaemia was 300 organisms per 100 microscope fields; during the first 90 days, peaks of parasitaemia occurred at intervals of 6-16 days and seldom exceeded 20 trypanosomes per 100 fields. Trypanosomes were only occasionally seen after day

786

CYCLICALLY TRANSMITTED TRYPANOSOME INFECTIONS IN CHIMPANZEES

91, and were last seen in a stained thick blood film 18 months after the infective fly bite: During the following 9 months, no trypanosomes were found, and observations were then discontinued.

Trypanosomes in the cerebrospinalfluid T. brucei had entered the cerebrospinal fluid by day 14, when 20 parasites per c.mm. were seen (Table I). By day 44, T. rhodesiense had invaded the central nervous system to the extent of 2 organisms per-c.mm. On day 65, one trypanosome was seen in 200 microscope fields (×40 objective) of a wet preparation of the cerebrospinal fluid from the chimpanzee infected with T. gambiense. On day 99, the cerebrospinal fluid was infective to a 12-day suckling rat although no trypanosomes were found by the direct examination of either the fluid or the blood. On 3 later occasions when cerebrospinal fluid was taken, no trypanosomes were seen, and no infections developed in 2-3 nursling rats inoculated each time. Body temperatures The pre-infection body temperatures in the chimpanzees ranged from 97.6 ° to 100"4°F. With all the infections there was a sharp increase in body temperature when the trypanosomes were first found in the blood. In the T. brucd infection the maximum temperature recorded was 102.4°F. on day 9 as parasitaemia was nearing its peak; when the chimpanzee was in extremis on day 14, its temperature was 97.8°F. In the T. rhodesiense infection, the highest temperatures of up to 102.4°F. were recorded the day before and during the first days of patent infection. During the remainder of the chimpanzee's life the body temperature ranged between 98.4 ° and 101.6°F.; the maximum temperatures did not appear to be associated with peaks of parasitaemia. The maximum body teml~erature of 102.2°F. observed in the infection with T. gambiense occurred on the first day of patency; thereafter the maximum recorded was 101.0°F. and, again, there appeared to be no association between the numbers or the appearance of trypanosomes and an increase in body temperature. Blood values Erythrocyte sedimentation rates. In each of the chimpanzees this rate increased during the course of the infection (Table II). The increase did not occur in the infection with T. gambiense until a long time after the other infections, and then not to such a marked degree. After administration of melarsen-oxide-BALto the chimpanzee infected with T. brucei the erythrocyte sedimentation rate returned almost to normal. Haemoglobin vodues. A very distinct decrease in haemoglobin occurred by day 44 in the chimpanzee infected with T. rhodesiense (Table II); with the T. bruce/infection the decrease was not so marked, and it was only slight at any time with the T. gambiense infection. Again, the pre-infection level was reached after cure of the chimpanzee infected with T. brucei. Erythrocyte count. The only marked decrease occurred by day 44 in the animal it~fected with T. rhoclesiense (Table II). The leucocyte count appeared to decrease in the early stages of infection (Table II).

787

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D. G. GODFREY AND R. KILLICK-KENDRICK

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Cerebrospinalfluid At times it was difficult to obtain sufficient, or indeed any, cerebrospinal fluid; hence the number of observations was not as great as we would have wished. Other than the presence of trypanosomes, the only observation made was on the numbers of cells. There was a distinct increase in the numbers of white cells during each infection (Table I). These numbers did not decrease when the chimpanzee infected with T. brucei was cured, but in the ape infected with T. gambiense the number of white cells fell to normal pre-infection levels after day 99. General condition The chimpanzee infected with T. brucei became obviously unwell on day 8 and, although on day 10 it appeared a little brighter, it became progressively worse, until by day 14 the animal was collapsed and seemed certain to die; about 1 "4 kg. had been lost in weight. The animal was suffering from severe diarrhoea and appeared to have a heavy infection with Strongyloides. On each of days 14, 16 and 17 1.9 ml. 3.6% w/v melarsen- oxide- BAL in propylene glycol was administered intravenously and the animal gradually recovered. The infection with T. rhodesiense did not appear to distress the chimpanzee until day 35 when sores developed on the gums and Sulphatriad (May and Baker) was given for 3 days. By day 41 the chimpanzee was very ill and lethargic. On day 44 it died while being anaesthetized for the routine examinations. Little change occurred in weight during the first month of the infection but between days 28 and 35, 1.0 kg. was lost. Post mortem examination was carried out by Mr. C. J. Roberts of this Institute. Large quantities of fluid containing trypanosomes were found in the pericardial sac and thoracic cavity. The pericardium was thickened and showed external adhesions of recent origin. The heart muscle was atonic. All lobes of the right lung were consolidated and this organ had obviously not been functioning for some time. The left lung was in reasonable condition. Death appeared to be due to overdosage with barbiturates, precipitated by the animal's weakness. The chimpanzee infected with T. gambieme did not appear to suffer unduly. During the very early days of the infection the swelling that developed on the leg at the site of the tsetse fly bite irritated the animal a little and until day 16 it was occasionally subdued; thereafter the infection caused no apparent distress. At the time of the infective feed the chimpanzee weighed 10.5 kg. and, with only minor fluctuations, it remained the same for 4 months; by day 252 the weight had increased to 12.6 kg. and the increase has since continued. Discussion

In these experiments with chimpanzees, T. brucei was the most pathogenic trypanosome. The chimpanzee infected with this organism would almost certainly have died 8 days after trypanosomes were first seen in the blood had it not been treated in extremis. The parasitaemia was high on most days of the infection and the trypanosomes soon invaded the central nervous system in large numbers. We have found only one report of an infection with T. bruc.a" in a chimpanzee. B~va~ (1962) inoculated infected blood into a chimpanzee and reports that when the blood was first examined 8 days later, a moderate parasitaemia was seen; he goes on to state that death occurred on the 12th day, from an intercurrem infection. Our chimpanzee was apparently about to die on the 14th day and we suggest that it is more likely that

790

CYCLICALLY TRANSMITTED TRYPANOSOME INFECTIONS IN CHIMPANZEES

Baker's chimpanzee died of acute trypanosomiasis with involvement of the central nervous system. Since the animal was inoculated with a very large number of trypanosomes (15 million), an initial high parasitaemia may have already been on the decline by the 8th day when the blood was first examined. BAKER(personal communication, 1964) agrees with us that this interpretation does not conflict with his experimental results. Baker planned his experiments in the hope that the chimpanzee resembled man in being resistant to infection with T. brucei but susceptible to T. rhodesiense; if this were so, chimpanzees could be used to distinguish these two species ot trypanosomes. Although his, and our, results prove that the chimpanzee is susceptible to both species it may still be possible to distinguish between them by their pathogenicity to this animal. In the present study, T. brucei was found to be extraordinarily pathogenic to a chimpanzee and we have suggested that Baker's experiment in fact had a similar result. Our chimpanzee infected with T. rhoclesiense lived 3 times as long as the one with T. brucei, and no apparent distress occurred during the first month; Baker also reports a very long chronic infection by T. rhodesiense, with no accompanying clinical illness. However, the apparently greater pathogenicity of T. brucei to the chimpanzee must be confirmed with several strains in a number of the animals before it can be accepted as a characteristic feature. The strains of T. brucei used by Baker and ourselves both came from Nigeria. Another factor which must be considered in assessing the pathogenicity of these two trypanosomes to chimpanzees is the effect of other disease. The ape with T. brucei had severe diarrhoea and helminthiasis, although in this case trypanosomiasis was obviously the main cause of distress as the cure was so dramatic. The position was not so clear in the animal with T. rhodesiense which had gross lung involvement, and the problem remains whether the major effect was due to trypanosomiasis, another disease, or both. The chronic infection with T. gambiense was similar to that reported by Tnotc~s and LnqTON (1904); a long chronic infection is also indicated in at least one case by Luc_~se (1964), who gives only a few details of his experiments. BAKER (1962) points OUt the possible usefulness of chimpanzees in the study of Rhodesian sleeping sickness because the central nervous system is involved. They may be useful for the Gambian disease as well, since the pathological changes observed by LucAss~, (1964) in one chimpanzee suggest penetration of the central nervous system, and the present experiment with T. gambiense confirms this observation. It is interesting that the isolation of T. gambiense used in our present experiments caused a very chronic disease and originated from Benue Province, an area noted for chronic human infections (DuGGA~, 1962). With the infections of T. rhodesiense and T. gambiense distinct swellings appeared at the site of bite soon after the flies had fed; in the T. brucei infection the swelling was neither as marked nor lasted as long. The swellings were probablythe sameasthe primary chancre of T. rhodesiense in a human volunteer described in detail by F~,IRaAntN and GOD}'I~Y (1957). Although the maximum size of the swelling in the chimpanzee infected with T. rhodesiense was smaller than that recorded in man, the developmental time was similar, and the maximum size ineach case was reached about the same time as trypanosomes were first found in the blood. The swelling in the chimpanzee with T. gambiense was larger than that reported by F,~J~,~a~I and GODFREY(1957) for the patient with T. rhodesiense. These observations again indicate the possible value of the chimpanzee for research into the human trypanosomiases.

D. G. GODFREY AND R. KILLICK-KENDRICK

791

Summary Trypanosoma brucei, T. rhodesiense and T. gambiense were transmitted by Glossina palpalis to 3 chimpanzees; the strain of T. brucei was shown to be non-infective to man. High parasitaemias developed in the chimpanzee infected with T. brucei, and 14 days after the infective fly had fed trypanosomes were seen in the central nervous system. At this stage the animal was acutely ill and the erythrocyte sedimentation rate had increased; there was only a slight fall in the haemoglobin value. The chimpanzee appeared to be about to die but was cured by treatment with melarsen-oxide-BAL. The infection with T. rhodesjense showed a fluctuating parasitaemia and had no apparent ill effect upon the chimpanzee for about one month after the infective feed. The general condition of the animal then deteriorated, and 44 days after infection the erythrocyte sedimentation rate had increased considerably and there was a marked anaemia; trypanosomes had invaded the central nervous system. The chimpanzee died while being anaesthetized. Large quantities of fluid containing trypanosomes were found post mortem in the pericardial sac and the thoracic cavity. The course of the infection with T. gambiense was mild, long lasting and did not appear to affect the chimpanzee unduly. The only noticeable change was an increase in the erythrocyte sedimentation rate. Trypanosomes were demonstrated in the cerebrospinal fluid on two occasions. The development of primary chancres and the invasion of the central nervous system by T. rhodesiense and T. gambiense emphasize the potential role of the chimpanzee in experimental studies on African trypanosomiasis. It is also suggested that the extremely virulent nature of T. brucei in the chimpanzee may be a means of distinguishing this species from T. rhodesiense, but more evidence is needed concerning other strains, host variations and the effect of intercurrent disease. REFERENCES BAKER, J. R. (1962). Ann. trop. Med. Parasit., 56, 216. BURTT, E. (1946). Ibid., 40, 141. Duc~&~r, A. J. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 439. FAmB~aRN, H. & BURTT,E. (1946). Ann. trop. Med. Parasit., 40, 270. & GODFREY,D. G. (1957). 1bid., 51, 464. H o ~ , C. A. (1964). J. ProtozooL, 11, 200. KXLLXCK-I~NDRICK,R. & GODFREY,D. G. (1963). Ann. trop. Med. Parasit., 57, 225. LUCASSE,C. (1964). Bull. Soc. Path. exot., 57, 283. THOMAS, H. W. & LINTON, S. F. (1904). Lancet, 1, 1337. WILLETT,K. (1956). Ann. trop. ivied. Parasit., 50, 75.