Sleeping sickness survey in Musoma district, Tanzania: Further study on the vector role of Glossina

403 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 68. No. 5. 1974.

SLEEPING SICKNESS SURVEY IN MUSOMA DISTRICT, TANZANIA: FURTHER STUDY ON THE VECTOR R()LE OF GLO$$1NA S. K. MOLOO* AND S. B. KUTUZA

East African Trypanosomiasis Research Organization, Tororo, Uganda Introduction

Trypanosoma (Trypanozoon) rhodesiense (HoARE, 1966) was allegedly introduced into Musoma District of Tanzania in the early 1920s by infected persons coming from Maswa, Mwanza District, where an epidemic of Rhodesian sleeping sickness had erupted in 1920 (FAIP,BAIRN, 1948). Glossina swynnertoni was the vector responsible for transmission of the disease (SwYNNERTON, 1923). The spread of the disease was caused either by infected people travelling between villages (SwYm~ERTON, 1923, 1925) or by game moving over large territories (DAVEY, 1924). In any case, this region remained an endemic focus of sleeping sickness and the transmission of the causative organism T. (T.) rhodesiense to man continued until 1954; thereafter the disease seemed to have disappeared, probably due to the closure of the gold mines in Kilimafedha and associated mines in the district with the resultant evacuation of the mining settlements, which considerably alleviated man-fly contact. The recrudescence of human trypanosomiasis in 1964 followed by progressive annual increase of cases could be attributed to the development of the two game sanctuaries, Serengeti National Park and Ikoma Game Reserve, and the concurrent increase in human population and their activities. The reappearance of the disease caused some concern particularly as this region had been developed as a tourist centre. Consequently, a large-scale survey of human trypanosomiasis in Musoma District in 1970 was carried out mainly in Serengeti National Park, Ikoma Game Reserve and a few villages neighbouring these game sanctuaries (O~ANGO and Woo, 1971 ; MOLOO et al., 1971; MWAMBU and MAYE~DE, 1971; GEIGY et al., 1971 ; ONYANGOet al., 1971). The aims of the investigation were to determine the mechanism and extent of transmission of sleeping sickness in this part of Tanzania. Although this survey revealed reservoirs of brucei-subgroup organisms in game (GEIGY et al., 1971) and in cattle (MwAMBU and MAYENDE, 1971) in the Ikoma-Serengeti region, the r61e of the local Glossina species in the biocenosis of disease still remained obscure. For example, MOLOO and his co-workers (1971) dissected 6,344 G. swynnertoni and 623 G. pallidipes but did not detect any salivary gland infection. In contrast, 10% of 115 wild mammals (GEIGY et al., 1971) and 3.5% of 798 cattle (MwAMBU and MAYENDE, 1971) were found infected with brucei-subgroup organisms. A year later, GEIGY and KAtWFMA~CN(1973) isolated further strains of brucei-subgroup from game in this endemic region of Tanzania; yet again no salivary gland infection was detected in any of the 3,550 G. swynnertoni dissected (ROGERSand BOREHAM 1973). Since the vector r61e of the local tsetse was not established despite these intensive investigations, a follow-up survey was undertaken in May and June 1972 in the same ecological zone but this time attention was concentrated on the incidence of brucei-subgroup infection in Glossina using the fly trituration method of LUMSDEN et al. (1963). The results are presented in this paper and discussed mainly in relation to the epidemiology of Rhodesian sleeping sickness. Materials and methods The 10 areas surveyed are shown on the map (Fig. 1). The first 6 were the same as those examined in 1970 (MoLoo et al., 1971); the remaining 4 were additional areas investigated within this IkomaSerengeti region. Initially, collection of the local tsetse was made using the following 3 methods of sampling: (i) Standard sample--a group of 4 field assistants caught flies with hand-nets on fly-rounds. *Present address: Tsetse Research Laboratory, Department of Veterinary Medicine, University of Bristol, Langford, Bristol BS18 7DU. We are grateful to the Director and staff of Tanzania National Park, and of the Serengeti Research Institute for their assistance during the survey. We also wish to thank Mr. D. P. T. Kahungu for technical assistance, the EATRO field staff for valuable help, and the Director of EATRO for permission to publish this paper.

404

SLEEPING SICKNESS SURVEY IN MUSOMA DISTRICT~ TANZANIA

(ii)

Trap sample--flies were caught in 2 traps, a Box-Screen trap (LANGRIDGE,unpublished) and an Awning Screen-Skirt trap (MOLOO, 1973).

(iii)

Vehicle sample~a Land Rover was driven through open woodland and along thickets, stopping at intervals to catch flies which had entered the vehicle and those that alighted on the body and the ground nearby. bou dO y MO=~ roads . . . . . . . . . . . Pork trocks . . . . . . . . . . . . . . . . . Dry slogan tr~cks. . . . . . . . . s........

Landing ground T i l t s = /urv¢ y

..............

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L."

.

1-

0

~

/ ZANt

Sabora I

u

S

Du~'wo

Plain

Ny

amumo

f.,

~:,~.

~

".,

i','~) I :~ I ~

k

u

,~"~-~

i ~'i l ~ ' ' " '

,~ , ~ '10

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3~0 K i l o m l t r e s t

Map of the Ikoma-Serengeti region of Tanzania showing locations of the tsetse survey areas.

G. swynnertoni and G. pallidipes were collected by these methods, the former species comprising the major proportion of the catch. The vehicle sampling method collected by far the largest numbers of both sexes of G. swynnertoni in a relatively short time. This method was therefore used exclusively in all the areas except Area 5 (adjacent to Ikoma Fort) where, in addition to a Land Rover, the above 2 traps were used since in 1970 G. pallidipes were caught in traps in markedly large numbers (MOLOO et al., 1971). However, this time only a few individuals of G. pallidipes were encountered. Hence, flies used in the present study were collected almost entirely by the vehicle sampling method. Flies were kept in Geigy-25 cages (NASH et al., 1966) or in 7"5 × 2-5 cm. tubes having mosquito netting at one end and cork at the other. These fly containers were kept covered with damp towelling in expanded polystyrene boxes in order to minimize death by desiccation, and thus transported to the Serengeti Research Institute laboratory in Banagi. The survivors were sorted by area, species and sex, and then grouped in batches of 50. Smaller batches were used when unavoidable. Each batch was anaesthetized with ether and triturated for about 3 min. with glass powder in a mortar to which a small quantity of borate buffer (pH 8.0) had been added. Further diluent was then added to give a final total of 1 ml. diluent per fly. The resultant suspension was poured into a test-tube and about 5 rain. later, 0.5 ml. of the supernate was inoculated intraperitoneally (IP) into each of 5 Swiss white mice which had previously been injected (IP) with 1 mg. of aqueous cyclophosphamide per mouse to suppress the immune response (WALKER, 1968); thereafter, this dose of cyclophosphamide was continued for 3 consecutive days. Starting on the third day after inoculation of the supernate, wet blood films were made daily from tails of the mice and examined to determine trypanosome infection. Thin blood films prepared from all parasitaemic mice were stained with Giemsa and the infecting trypanosome types identified. The brucei-subgroup strains isolated were preserved in liquid nitrogen (DAR et al., 1972) and stored in WHO/EATRO bank. The

405

S. K. M O L O O AND S. B. K U T U Z A

congolense-group infections when encountered were recorded and the infected mice were subsequently discarded. T h e aparasitaemic mice were examined daily for 40 days after inoculation and then killed. The brucei-subgroup strains isolated were later tested in EATRO by the Blood Incubation Infectivity Test (BIIT) of RIcKMAN and ROBSON(1970) to differentiate between T. (T.) brucei and T. (T.) rhodesiense. 2 capillaries of each brucei-subgroup stabilate were diluted with 1.5 ml. of phosphate-buffered saline (pH 7.4) and then 0-4 ml. was injected (IP) into each of 2 mature rats. When parasitaemias varying from -k 4- to q- -k q- were observed on wet blood film examinations, the respective donor rat was bled from the heart and 0-25 ml. added to 2 ml. of oxalated fresh human blood in a 6 × 1"5 cm. tube. This mixture was incubated in a water-bath at 37°C. for 5 hr. after which 0.5 ml. was inoculated (IP) into each of 2 rats. The rats were examined daily by preparing wet blood films from their tails for 40 days, or until they showed persistent parasitaemia, and then killed. Each of the 9 brucei-subgroup strains was tested simultaneously with 2 similarly treated standards, T. (T.) rhodesiense (EATRO 1825 or its derivative EATRO 2059) and T. (T.) brucei (EATRO 1822 or its derivative EATRO 2005), and a phosphate-buffered-glucose saline (pH 7.4) control. The 3 replicates carried out on each of these were tested concurrently. T. (T.) rhodesiense (EATRO 1825/2059) and T. (T.) brucei (EATRO 1822/2005) strains used as standards were originally isolated respectively from a cow, (MWAMBUand MAYENDE,1971) and a lion (GEIGY et al., 1971), and tested in human volunteers by DR. R. J. OYANGO (GEIGY et al., 1973). Results Table I shows the number of males and females of G. swynnertoni and G. pallidipes examined from the 10 different areas within the Ikoma-Serengeti region, and the number of brucei-subgroup strains isolated. It should be noted that in calculation of the infection rates it was assumed that only one fly in a batch that produced infection in mice was infected; hence the infection rate figures given in the Table are minimal. Out of 128 batches of G. swynnertoni males examined (6,371 flies), 5 brucei-subgroup strains were isolated (infection rate, 0.078%); 2 from an area between Banagi and Retima Hippo Pool, and 3 from Muswiranyoko Valley. Examination of 94 batches of females (4,689 flies) showed 4 such infections (0-085%) all from an area in the neighbourhood of Ikoma Gate. Altogether 222 batches of G. swynnertoni (11,060 flies) were examined from which 9 brucei-subgroup strains were isolated, giving an infection rate of 0.081 ~/o The number of G. pallidipes caught was too small. In all 4 batches comprising 95 flies were examined but none revealed brucei-subgroup infection. TABLE I. T h e numbers of G. swynnertoni and G. pallidipes examined from 10 different areas and the incidence of brucei-subgroup infection Males

Study areas

Species

Number in a batch

Number of batches

Females

Total

Infection

Number in a batch

Number of batches

Total

Infection

Total flies examined

1 Between Banagi and Retima H i p p o Pool

G. swynnertoni

50 21

16 1

821

2 (0.24%)

50 39

3 1

189

0

1010

2 Between Banagi and Seronera

G. swynnertoni

50

13

650

0

50

8

400

0

1050

3 Kilimefedha

G. swynnertoni

50

12

600

0

50

8

400

0

1000

4 Around Ikoma Gate

G. swynnertoni

50

12

600

0

50

8

400

4

1000

5 Adjacent to Fort Ikoma

G. swynnertoni G. pallidipes

50 13

24 1

1200 13

0 0

50 32

16 1

800 32

0 0

2000 45

6 Close to Retima

G. swynnertoni

50

10

500

0

50

10

500

0

1000

7 Muswiranyoko Valley

G. swynnertoni

50

10

500

10

500

0

1000

G. swynnertoni

50

10

500

3 (0'6%) 0

50

8 Close to Kamarishe hills

50

10

500

0

1000

9 Close to Simiti and M u m u g h i a hills

G. swynnertoni G. pallidipes

50 38

10 1

500 38

0 0

50 12

10 1

500 12

0 0

1000 50

G. swynnertonl

50

10

500

0

50

I0

500

0

1000

Hippo Pool

10 Togoro Plain

(1.0%)

Table I I shows the results of B I I tests carried out on the 9 brucei-subgroup isolates (designated stabilates E A T R O 2011-2019), the two standards, namely T. (T.) rhodesiense and T. (T.) brucei, and the controls. All the 9 brucei-subgroup strains gave consistently negative results whereas all the controls

SLEEPING SICKNESS SURVEY IN MUSOMA DISTRICT, TANZANIA

406

resulted consistently The

BII

in positive results. However,

tests on this standard

gave 2 positive

and 2 equivocal

results (batches

the BII tests on

the T. (T.) brucei s t a n d a r d

2 tests gave negative TABLE I I . B I I T

and

the

T. (T.) rhodesiense s t a n d a r d

(batches

1 and

3 and 6, each gave 2 negative resulted

8), 5 negative and

consistently

behaved

quite abnormally.

(batches

2, 4, 5, 7 and 9),

1 positive results). On the other hand, in negative

except for batch

6 in which

1 positive results.

results of the 9

brucei-subgroup ( E A T R O Ikoma-Serengeti

2011-2019) area of Tanzania.

isolated from

G. swynnertoni in the

Results Batch

4

T C S1 S2

= = = =

Test. Control. Standard Standard

EATRO

stabilates

1

2

3

2011T 2011C 1825 S 1 1822 S 2

+ + --

+ + -

-+ + -

2012T 2012 C 1825 S 1 1822 S 2

-+ -

+ -

+ -

2013T 2013 C 2059 S 1 2005 S 2

-+ --

-+ + -

-+ --

2014T 2014 C 2059 S 1 2005 S 2

-+ ---

+ ---

+ --

2015T 2015 C 2059 S 1 2005 S ~

-+ --

+ ---

-+ --

2016T 2016 C 2059 S 1 2005 S 2

+ + -

+ -

+ +

2017T 2017 C 2059 S 1 2005 S 2

-+ -

-+ --

+ --

2018T 2018 C 2059 S 1 2005 S 2

-+ + --

-+ + --

-+ + --

2019T 2019 C 2059 S 1 2005 S 2

+ --

+ --

+ ---

T. (T.) rhodesiense ( E A T R O T. (T.) brucei ( E A T R O 1 8 2 2

--

1825 or its derivative EATRO 2059). or its derivative EATRO 2005).

Discussion

re#on

I n t h i s s u r v e y 9 brucei-subgroup s t r a i n s w e r e i s o l a t e d f r o m G. swynnertoni f r o m t h e I k o m a - S e r e n g e t i of Tanzania. The trituration method coupled with the use in mice of the immuno-suppressant,

S. K. M O L O 0 AND S. B. KUTUZA

407

cyclophosphamide, would seem to be more efficient for determining brucei-subgroup infections in Glossina than the dissection method which, in the 1970 survey of the same region (MOLOO et al., 1971) and again in the 1971 survey (ROGERS and BOREHAM, 1973), did not reveal any such infection in 9,894 G. swynnertoni examined. In both these previous surveys the salivary glands of tsetses were examined thoroughly, so that it is improbable that some mature brucei-subgroup infections were present but all with such scant parasitaemias in the glands that they remained undetected. Unless none of these tsetses was carrying such infection, and hence the lack of trypanosomes in the salivary glands, the more plausible explanation would seem to be that those tsetses with mature brucei-subgroup infections the metatrypanosomes were located in organ (or organs) other than these glands. Recent laboratory work suggests that this could have been the case. For example, MSHELBWALA(1972) found metatrypanosomes in the haemocoel of tsetses with not only mature but also immature T. (T.) brucei infection. It is indeed noteworthy that inoculation (IP) into a mouse of the infected haemolymph from a T. (T.) brucei infected tsetse showing positive midgut and heamocoel but negative salivary glands and proboscis, resulted in infection. Whether such tsetses can transmit infection to their hosts during feeding is unknown. However, WARD and BELL (1971 reported that when tsetses infected with T. (T.) brucei were individually fed on mice, the transmission rate was some 5 times higher than revealed by salivary gland examination. These findings evidently do not provide conclusive evidence that transmission of brucei-subgroup may be effected by infected tsetse lacking the salivary gland infection, but rather they illustrate the need for reinvestigation of the life-cycle of bruceisubgroup in Glossina and for evaluation of the relative efficiency of different methods of determining such infections in this vector. Nevertheless, the present survey has established the vector r61e of G. swynnertoni in the transmission cycle of brucei-subgroup organisms in the Ikoma-Serengeti region. All the 9 brucei-subgroup strains were BIIT-negative while the respective controls were positive. It would appear therefore that all these strains are T. (T.) brucei. However, the B I I T results of the standards suggest that this might not be a valid interpretation. For example, whereas on T. (T.) brucei (EATRO 1822/2005) the BII tests were negative in 8 batches and equivocal in 1, on T. (T.) rhodesiense (EATRO 1825/2059) the results were markedly inconsistent. Only in 2 batches were the results positive, and of the remaining 7 batches 5 were negative and 2 equivocal. It should be noted that of the total of 27 replicate tests carried out on the T. (T.) rhodesiense standard, only 8 resulted in positive (29.6%) and the remaining 19 in negative (70-4%). It seems therefore that the BII test in its present form is not reliable for differentiating between T. (T.) rhodesiense and T. (T.) brucei. Investigation to find out what this test actually measures should be a useful prerequisite to its better understanding and its improvement. Nevertheless, the testing of strains in volunteers for their pathogenicity to man is at present the only reliable method of differentiating between these two trypanosome species, and since this was not carried out the 9 brucei-subgroup isolates could not be classified further. In the Ikoma-Serengeti region the rate of brucei-subgroup infection in G. swynnertoni was 0.08%. In endemic areas low brucei-subgroup infection rate in tsetse is indeed a known fact. For example, in a Rhodesian sleeping sickness area in Tanzania, VANDERVLANK(1947) dissected 35,112 G. swynnertoni and G. pallidipes; infections attributed to brucei-subgroup were under 0.1%. In Mara region, Kenya, the corresponding figures were 0"08~o in 5,928 G. swynnertoni examined (WILSON et al., 1972). During an outbreak due to T. (T.) rhodesiense in the Lake Province, Tanzania, DUKE (1923) dissected 2,206 G. swynnertoni collected from 3 types of locality and found the following rates of brucei-subgroup infection: (i) 0-24% of 819 flies from the locality where the inhabitants were heavily infected. (ii) 0-1% of 722 flies from villages where the inhabitants were less heavily infected. (iii) None of 665 flies collected from the uninhabited locality was carrying salivary gland infection. Despite low infection rates intense transmission may still occur in endemic areas. The brucei-subgroup infections in G. swynnertoni were encountered from only 3 of the 10 areas examined. These were (i) an area between Banagi and Retima Hippo Pool (Infection rate >~0-2%); Muswiranyoko Valley (/> 0.3%); and (ii) an area in the neighbourhood of Ikoma Gate (>/0.4%). BanagiHippo Pool region normally contains a variety of game species, including hippopotamus and crocodile in the pool, and is therefore frequented by tourists as well as staff of the Serengeti National Park (SNP). The other two areas also are good for game watching and, in addition, the SNP staff who are posted at the Ikoma Gate are constantly exposed to tsetse attacks, and at the time of the survey there were some two dozen men in Muswiranyoko Valley engaged in drilling a borehole for a permanent supply of water. Such human encroachment of the heavily infested tsetse bush evidently enhances contact with G. swynnertoni which is not only readily adaptable in its feeding habit but would feed from man (MoLoo et al., 1971).

408

SLEEPING SICKNESS SURVEY IN MUSOMA DISTRICT, TANZANIA

It is noteworthy that many strains of brucei-subgroup were isolated from a limited number of game species examined from various parts of the park, including Banagi-Hippo Pool region, Ikoma Gate and their contiguous areas (GEIGY and KAUFFMANN, 1973). Also, since the reappearance of Rhodesian sleeping sickness in 1964 many of the cases in the Ikoma-Serengcti region allegedly contracted the infection from the above three areas. Yet these areas can not be considered as restricted loci of sleeping sickness infection for the following reasons. Firstly, many game species move extensively over large areas of the park, and the available data on game show no significant differences in the incidence of brucei-subgroup infection between different areas within the park, even in the case of relatively resident species such as hartebeest (BERTRAM, 1973). Secondly, the bmce{-subgroup infection rate in tsetse is normally very low, so that the size of the sample examined in each of the 10 areas was too small to postulate infection loci in areas where infected Glossina were encountered. Besides, populations of tsetse are far from static and, as in the case of game, it is unlikely that the brucei-subgroup infection rates in this vector are very much different from one area of the Ikoma-Serengcti region to another. Thirdly, cases of Rhodesian sleeping sickness have also been notified from some of the other areas where Glossina were found lacking in brucei-subgroup infection. In the Ikoma-Screngeti region therefore there are no restricted loci of sleeping sickness infection but rather the various components comprising zoonosis interact sporadically throughout this ecological zone. The reservoir of T. (T.) rhodesiense is in the game animals which evidently disperse the pathogen as they range extensively over large territory. G. swynnertoni is abundant almost throughout this region and feeds on a wide range of vertebrate hosts including bovids, suids, elephants, hippopotami, primates, carnivores, aardvark and avians (MoLoo et al., 1971; MOLOO, 1973). G. pallidipes and G. brevipalpis are also present but have restricted distribution. G. pallidipes is in the main confined to drainage vegetation whereas G. brevipalpis to areas with dense thickets (MoLoo et al., 1971). The Ikoma-Serengeti region supports an abundance of game and tsetses, and consequently there is intense circulation of the parasites, via the vector, among susceptible individuals of the same as well as different host species. However in the case of lions and hyaenas which are heavily infected (BAKER, 1968; GEIGY and KAtWFMANlq, 1973) it is possible that the following transmission mechanisms are operative. Transmission of infection by feeding on infected carcases has been demonstrated (BRucE, 1897; DUKE et al., 1934; MOLOO et al., 1973). This oral route of transmission is possibly common in nature among the above carnivores which live in areas where a proportion of their prey are infected (MoLoo et al., 1973). The spread of infection may occur among members of the same group of lions in the course of extensive social grooming when they lick one another's wounds (BERTRAM,1973). Also, carnivores are occasionally fed from by G. swynnertoni (MoLOO et al., 1971), so that transmission is possibly also effected, albeit to a limited extent, by the cyclical mechanism. Although lions and hyaenas are frequently infected, they may be less significant in the endemicity of Rhodesian sleeping sickness compared with the vastly abundant, less infected and less studied game species. In any case, the disease is maintained in this region as a zoonotic infection. People have been infected when they encroached onto the tsetse bush and encountered infected flies. The incidence of the disease has thus depended on many factors, the major one being man-fly contact. The progressive increase in population which followed the development of the tourist industry in this region promoted paripassu man-fly contact during which infection with T. (T.) rhodesiense may be contracted by susceptible persons. This could explain the progressive increase of cases since its reappearance in 1964. However, the sporadic occurrence of the disease is suggestive of the restricted nature of man-fly contact in this endemic region. The abundance of game evidently serves as a valuable buffer between man and the tsetse vector, and in the present situation preserves him from more intense attack. In this part of Tanzania, therefore, the biocenosis of Rhodesian sleeping sickness favours a continuous transmission of the parasites, via tsetses, among game animals some of which function as natural reservoirs. In this ecological zone man is only an incidental host. Summary In a follow-up survey of sleeping sickness in the Ikoma-Serengeti region of Tanzania, carried out in May and June 1972, 11,060 G. swynnertoni and 95 G. pallidipes were collected, triturated mainly in batches of 50, and the supernate of each batch was inoculated into cyclophosphamide-treated mice. Nine strains of brucei-subgroup were isolated from G. swynnertoni, which indicated the vector r61e of the local tsetses in the transmission cycle of infection in this endemic region.

8. K. MOLOOAND S. B. KIJUTZA

409

All the 9 brucei-subgroup isolates gave consistently negative results by the Blood Incubation Infectivity Test (BIIT) whereas the respective controls gave positive results. However, T. (T.) brucei and particularly T. (T.) rhodesiense standards which were tested simultaneously with the test strains gave inconsistent results. It would seem therefore that the BII test in its present form is not reliable for differentiating between the above two trypanosome species, and so the 9 brucei-subgroup isolates could not be classified further. In the Ikoma-Serengeti region there are no restricted loci of Rhodesian sleeping sickness but rather the various components comprising zoonosis interact sporadically throughout this part of Tanzania. The biocenosis of infection favours intense circulation of the parasites, mainly via the tsetse vectors, among game animals some of which function as natural reservoirs; in this ecological zone man is only an incidental host. REFERENCES BAKER, J. R. (1968). Syrup. zool. Soc. Lond., 24, 147. BERTRAM, B. C. R. (1973). Acta trop., 30, 36. BRUCE, D. (1897). Further Report on the Tsetse Fly Disease or Nagana in Zululand, Umbobo, May, 1896. London: Harrison and Sons. DAR, F. K., LIGTHART,G. S. & WILSON, A. J. (1972). ft. Protozool., 19, 494. DAVEY, J. B. (1924). Trans. R. Soc. trop. Med. Hyg., 17, 474. DUKE, H. L. (1923). Proc. Roy. Soc. (B), 94, 250. , METTAM, R. W. M. & WALLACE,J. M. (1934). Trans. R. Soc. trop. Med. Hyg., 28, 77. FAIRBAIRN, H. (1948). Trop. Dis. Bull., 45, 1. GEIGY, R. & KAUFFMANN,M. (1973). Acta trop., 30, 12. , - - , MAYENDE, J. S. P., MWAMBU, P. M. & ONYANGO, R. J. (1973). Ibid., 30, 49. ~ , MWAMBU, P. M. & KAUFFMANN,M. (1971). Ibid., 28, 211. HOARE, C. A. (1966). Ergebn. Mikrobiol., 30, 43. LUMSDEN, W. H. R. ET AL. (1963). E A T R O Annual Report for 1961, p. 41. MoLoo, S. K. (1973). Ann. trop. Med. Parasit., 67, 205. (1973). Bull. ent. Res., 63, 231. ~ , Losos, G. J. & KUTUZA,S. B. (1973). Ann. trop. Med. Parasit., 67, 331. ~ ' , STEIGER,R. F., BRUN, R. & BOREHAM,P. F. L. (1971). Acta trop., 28, 189. MSHELBWALA,A. S. (1972). Trans. R. Soe. trop. Med. Hyg., 66, 637. MWAMBU, P. M. & MAYENDE, J. S. P. (1971). Acta trop., 28, 206. NASH, T. A. M., JORDAN,A. M. & BOYLE, J. A. (1966). Ann. trop. Med. Parasit., 60, 469. ONYANGO, R. J., GEIGY, R., MWAMBU, P. M. & MOLOO, S. K. (1971). Acta trop., 28, 221. & Woo, P. (1971). 1bid., 28, 181. RICKMAN, L. R. & ROBSON, J. (1970). Bull. Wld Hlth Org., 42, 911. ROGERS, D. & BOREHAM,P. F. L. (1973). Acta trop., 30, 24. SWYNNERTON, C. F. M. (1923). Bull. ent. Res., 13, 317. (1925). Trans. R. Soc. trop. Med. Hyg., 19, 70. VANDERPLANK,F. L. (1947). Ann. trop. Med. Parasit., 41, 365. WALKER, P. J. (1968). ft. Protozool., 15 (Suppl.), 33. WARD, R. A. & BELL, L. H. (1971). Trans. R. Soc. trop. Med. Hyg., 65, 236. WILSON, A. J., DAR, F. K. & PARIS, J. (1972). Trop. Anirn. Hlth. Prod., 4, 14.