EXPERIMENTAL PARASITOLOGY 72, 430-439 (191)

Tvpanosoma brucei rhodesiense: Characterisation of Stocks from Zambia, Kenya, and Uganda Using Repetitive DNA Probes

GEOFF HIDE,* NORMA BUCHANAN,* SUE WELBURN,~ IAN MAUDLIN,? J. DAVID BARRY* AND ANDREW TAIT*

* Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow University, Bearsden Road, Glasgow G61 1QH United Kingdom; fTsetse Research Laboratory, ODAIUniversity of

Bristol, Langford House, Langford, Bristol BS18 7DU United Kingdom; and SWellcome Unit of Molecular Parasitology, Department of Genetics, Glasgow University, Church Street,

Glasgow GlI 5JS United Kingdom

HIDE, G., BUCHANAN, N., WELBURN, S., MAUDLIN, I., BARRY, D., AND TAIT, A. 1991. Trypanosoma brucei rhodesiense: Characterisation of stocks from Zambia, Kenya, and Uganda using repetitive DNA probes. Experimental Parasitology 72, 43ti39. We have previously described a system for characterising the relationships between trypanosome stocks of the T.brucei group based on Southern blotting with repetitive DNA probes fol- lowed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Par- asitol. 39,213-226, 1990). In this study, we extend this analysis to examine the relationships between trypanosome stocks isolated from major sleeping sickness foci in Zambia, Kenya, and Uganda. We show that the trypanosome strains responsible for disease in Zambia are quite distinct from those sampled from the Kenya/Uganda foci. Furthermore, the human serum resistant stocks isolated from the Kenya/Uganda foci which were isolated from man (or from animals) were found to form a tight group in the cluster analysis, while stocks isolated from nonhuman sources in the same area or stocks from elsewhere were found in separate groups. Thus, the human infective trypanosome strains found in these foci may have common origins and have, perhaps, arisen by clonal selection from a common source. 0 1991 kademi~ press, Inc.

INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma brucei brucei; T.b.rhodesiense; T.b.gambiense; Kinetoplastida; Protozoa; Repetitive DNA; Ribosomal RNA genes; Molec- ular epidemiology; Strain characterisation; Epidemiology; Zambia; Kenya; Uganda; Cluster analysis; Southern blotting; Deoxyribonucleic acid (DNA); Ribonucleic acid (RNA); So- dium dodecyl sulphate (SDS).

INTRODUCTION

In a previous study, we developed a sys- tem for analysing the group relationships between stocks of African trypanosomes, of the Trypanosoma brucei group, based on variation in repetitive DNA sequences (Hide et al. 1990). Restriction fragment length polymorphisms, found within the trypanosome ribosomal RNA genes and a trypanosome-repetitive DNA sequence, generated a series of banding patterns, on Southern blots, which were characteristic for each trypanosome stock examined. Similarities and differences in the banding patterns, observed in different trypano- some stocks, were used to characterise the

degree of relatedness between the stocks using a cluster analysis technique. This ap- proach has been used here as a tool for ex- amining the relationships and epidemiology of Trypanosoma brucei rhodesiense stocks from Kenya, Uganda, and Zambia.

The first recorded cases of sleeping sick- ness caused by Ttypanosoma brucei rho- desiense were found in the Luangwa Valley of Zambia (Stephens and Fantham 1910). From the epidemic there, it is thought that T,b.rhodesiense spread northward, and by the 1940’s the important foci in Uganda and Kenya were established (Ormerod 1961). However, numerous differences have been observed, in the prevalence (Dukes et al. 1983, 1984) and virulence (MacKichan

430 0014-4894191 $3.00 Copyright 6 1991 by Academic Press, Inc. All rigbts of reproduction in any fom reserved.

CHARACTERISATION OF T.b.rhodesiense STOCKS 431

1944; Foulkes 1970; Buyst 1974; Rickman 1974) of T.b.rhodesiense-associated sleep- ing sickness between the major disease foci in Kenya/Uganda and those in Zambia. We have set out to investigate the epidemiolog- ical relationships between T.b.rhodesiense stocks isolated from these major foci in Kenya, Uganda, and Zambia using the re- petitive DNA system.

MATERIALS ANDMETHODS

Trypanosome stocks. The trypanosome stocks ex- amined in this study are described in Table I and in Hide et al. (1990).

Isolation of trypanosome DNA, plasmid, and phage DNA probes. Isolation of DNA from trypanosomes and DNA probes (lambda 104, lambda 109, and pBE2) was carried out as previously described (Hide 1988; Hide et al. 1990).

Southern blotting. Southern blotting was carried out as previously described (Hide et al. 1990) except that DNA was transferred on to Nylon filters (Amersham Hybond-N), instead of nitrocellulose, and the DNA was fixed to the filter by cross-linking by exposure to long-wave ultraviolet light for 7 min. The filter was prehybridised for 30 min in 0.5 M phosphate buffer, pH 7.2, 7% SDS at 65°C and hybridised in IO’ cpm of the appropriate DNA probe overnight at 65°C in 0.5 M phosphate buffer, pH 7.2, 7% SDS and washed twice for 15 min each wash in 0.1% SDS, 2 x SSC (Sarn- brook et al. 1989) at 65°C. These modifications of the Southern hybridisation method gave identical results to the previously described method (Hide et al. 1990).

Cluster analysis. Cluster analysis was carried out as previously described (Hide et nl. 1990) except that computer programs were used to construct the simi- larity matrices (BIGSIM) and dendrograms (4M). BIGSIM was a modification of the program SIM (writ- ten by R. E. Cibulskis, University of Liverpool) to handle larger data sets.

RESULTS

DNA sequences representing the riboso- ma1 RNA genes of T.brucei were used to probe Southern blots of trypanosome DNA from a number of T.b.rhodesiense stocks isolated from the Zambian, Kenyan, and Ugandan foci. The 40 stocks used in this analysis are listed in Table I and the results obtained from the analysis were combined

with another 50 stocks taken from a previ- ous analysis (Hide et al. 1990). The probes used include Lambda 104, a probe which represents the ribosomal coding region, Lambda 109, which represents the nontran- scribed spacer region, and pBE2 an unchar- acterised repetitive DNA sequence from trypanosomes (Hide et al. 1990). A typical Southern blot is shown in Fig. 1 and the banding pattern data obtained from a num- ber of such blots is presented in Figs. 2-5. The four sets of data obtained from the Southern analysis are as follows: Hind111 digests of trypanosome DNA probed with Lambda 104 (Fig. 2), Lambda 109 (Fig. 3), and pBE2 (Fig. 4) and XhoI digested try- panosome DNA probed with Lambda 109 (Fig. 5).

Cluster analysis was applied to the data on banding patterns obtained from the Southern blotting to determine the related- ness of each stock based on the similarity and differences between banding patterns. The results are shown in the dendrogram illustrated in Fig. 6. From the dendrogram it can be seen that nine distinct groups of T.brucei stocks are formed. A summary of the composition of each group of stocks is given below:

Group 1. This group contains a single hu- man-infective stock, 2208, which was iso- lated from the village of Kasyasya in Zam- bia.

Group 2. This group comprises three stocks isolated from tsetse in the Kiboko region of Kenya.

Group 3. Two stocks from West Africa, one of which was isolated from man and the other from cattle, are contained within this group. These stocks represent the West Af- rican “rhodesiense’‘-like trypanosomes.

Group 4. This group contains the T.b. gambiense stocks.

Group 5. This group comprises, exclu- sively, human-infective stocks isolated from Zambia.

Group 6. The stocks in this group are

432 HIDE ET AL.

TABLE I Trypano~omn brucei stocks used in this analysis

Stock Other designation Host Date Location

Kenyan stocks GUP793 GUPlo42 GUPlo43 GUPlOSl GUP1052 GUP2077 GUP2078 EATR0156 EATR0795 GUP2498

GPAL/KE/6lIEATROl8 Tsetse 1%1 Yimbo, Kenya MHOM/KE/6l/EATR094 MElll 1%1 Yimbo, Kenya MHOM/KE/6l/EATR095 Man 1%1 Yimbo, Kenya MHOM/KE/6l/EATRO% Man 1961 Yimbo, Kenya MHOM/KE/6l/EATRO97 Man 1961 Yimbo, Kenya MHOM/KE/6l/EATR0106 Man 1%1 Yimbo, Kenya MHOM/KEMl/EATROll6 Mall 1961 Yimbo, Kenya MHOM/KE/6l/EATROl56 Man 1961 Yimbo, Kenya MBOI/KE/ti/EATR0795 Cattle 1964 Alego, Kenya MHOM/KE/77iEATR02340 Man 1977 Samia, Kenya

Ugandan stocks GUPl075 GUPl301 UTRO2 UTR03 uTRo4 UGl UG2 UG3 UG4 UG5 UG6 UG7 UG8 UG9 UGlO UGll UG12 UG13 UG14 UGl5 UG16 UG17

MHOM/UG/59/EATR0174 Man 1959 Busoga, Uganda GPAL/UG/6O/EATR03 Tsetse 1960 Busoga, Uganda MHOM/UGI8lIUTRO2 Man 1981 Busoga, Uganda MHOM!UG/8l/UTR03 Man 1981 Busoga, Uganda MHOM/UG/8l/UTR04 Man 1982 Busoga, Uganda MBOYUG/88/IYOLWA96 Cattle 1988 Iyolwa, Uganda MHOM/UG/BS/UGANDA A Man 1988 Iyolwa, Uganda MHOMIUGI88IUGANDA F Man 1988 Iyolwa, Uganda MBOYUG/88/IYOLWAl25 Cattle 1988 Iyolwa, Uganda MBOIfLJG/SS/MELA27 Cattle 1988 Iyolwa, Uganda MBOI/UG/88/IYOLWAl02 Cattle 1988 Iyolwa, Uganda MHOM/UG/88/UGANDA H Man 1988 Iyolwa, Uganda MBOI/UG/88/MELA71 Cattle 1988 Iyolwa, Uganda MBOYUG/88/PAPOL42 Cattle 1988 Iyolwa, Uganda MSUS/UG/88/MELA PIG m 1988 Iyolwa, Uganda MBOYUG/88/MELA2 Cattle 1988 Iyolwa, Uganda MBOI/UG/88/POYEMll Cattle 1988 Iyolwa, Uganda MBOVUG/88/IYOLWAl53 Cattle 1988 Iyolwa, Uganda MHOM/UG/88/UGANDA B Man 1988 Iyolwa, Uganda MHOM/UG/88/UGANDA C Man 1988 Iyolwa, Uganda MHOMIUGI88IUGANDA E Man 1988 Iyolwa, Uganda MHOM/UG/88/UGANDA G Man 1988 Iyolwa, Uganda

Zambian stocks 2194 2208 2210 2218 2222 2269 2274 2290

MHOMIZM/8UZl94 Man 1982 Chibale, Zambia MHOM/ZM/82/Z208 Man 1982 Kasyasya, Zambia MHOM/ZM/82/Z210 Man 1982 Kasyasya, Zambia MHOM/ZM/82/Z218 Man 1982 Chibale, Zambia MHOMlZMl82JZ222 MkUl 1982 Kasyasya, Zambia MCAPlZMl83iZ269 Goat 1983 Kasyasya, Zambia MHOM/ZM/82/Z274 Man 1982 Kasyasya, Zambia MHOM/ZM/8l/Z% Man 1981 Chibale, Zambia

from West Africa and were isolated both number of stocks isolated from animals from humans and animals. As with group 3, (cattle and sheep), tsetse, and man from these stocks represent West African Kenya and Uganda. “rhodesiense’‘-like trypanosomes. Group 8. The stocks contained within

Group 7. This group contains a large this group are all isolated from cattle in

CHARACTERISATION OF T. b.rhodesiense STOCKS 433

A B

12345 123456

FIG. 1. A typical Southern blot showing banding patterns produced when Hind111 digested trypano- some DNA was probed with pBE2. (A) A comparison of Kenyan T.brucei stocks, TREU 869 (lane l), EATRO 156 (lane 3), GUP 1043 (lane 4), and stocks isolated from Zambia, Z 290 (lane 2) and Z 269 (lane 5). (B) A comparison of trypanosome stocks from Uganda. UG 14, 15, 16, and 17 (lanes 3-6, respec- tively) were isolated from man, and UG 12 and 13 (lanes 1 and 2, respectively) were human serum sen- sitive trypanosomes isolated from animals. Sizes of some of the bands are given in kilobases for reference purposes.

Uganda and are human serum sensitive (i.e., not human infective).

Group 9. The stocks contained within this group are isolated from pigs and cattle in Uganda and are human serum sensitive.

DISCUSSION

In a previous study, we have used a sys-

tem, based on similarities and differences in banding patterns generated by repetitive DNA probes, to characterise the relation- ships between trypanosome stocks (Hide et al. 1990). The main advantage of this sys- tem is that it is able to establish levels of identity and difference between trypano- some stocks, based on variation in multiple loci, without dependence on other biologi- cal information available on the stock. The sensitivity of this approach is enhanced by the use of a large number of characters (bands) such that high degrees of similarity in banding patterns reflect close genetic identity. It is not possible to place exact probabalistic values on the confidence with which genetic identity and difference can be established, using this approach, as no information is available on rates of change of these banding patterns in trypanosome field isolates. However, the sensitivity of this approach was assessed by a compari- son, carried out blind, with isoenzyme and biological characteristics obtained from other work (Maudlin et al., in preparation), using a collection of trypanosome stocks from Uganda (UGl-17). Complete agree- ment between these different approaches was observed. This system is, therefore, capable of answering a number of impor- tant epidemiological questions which may not be answerable using other approaches.

1 2 3 4 5 6 7 6 910111213141516171819 202122232425262726293031323334353637363940 ----------------------------------------,a ----------------I-----------------------~~ _____m-e--v-------------------- - ----80 ,,,,,1,,-11------,,-,,,,,,,,,1,,,,,,,,,,~~ ___________--_------------ - ----- -----so

- - - - _--- -------------- -- - -- - ----2: -- -- - - -- - 50

- - - ---- - -s--w - - :z

-- - - -- -- _--- - -w-m- - - - - - -- - ----j;

-- -- -- 35 _--- - -w-m- - - - -----------e-----35

-- _- -- -- - - - - - B,"

FIG. 2. Banding patterns observed for each of the trypanosome stocks examined in this study following Southern blotting of Hind111 restriction-digested trypanosome DNA and probing with Lambda 104. The trypanosome stocks are as follows: (1) 2222, (2) 2290, (3) EATRO 156, (4) GUP 1043, (5) GUP 2078, (6) GUP 2077, (7) 2194, (8) 2218, (9) EATRO 795, (10) 2269, (11) 2274, (12) GUP 1042, (13) GUP 1051, (14) UTRO 3, (15) GUP 1301, (16) UTRO 4, (17) 2210, (18) GUP 2498, (19) 2208, (20) GUP 1052, (21) UTRO 2, (22) GUP 1075, (23) GUP 793, (24) UGl, (25) UG2, (26) UG3, (27) UG4, (28) UGS, (29) UG6, (30) UG7, (31) UG8, (32) UG9, (33) UGlO, (34) UGll, (35) UG12, (36) UG13, (37) UG14, (38) UG15, (39) UG16, (40) UG17. Sizes of bands are indicated in kilobases.

434 HIDE ET AL.

1 2 3 4 5 6 7 8 91011121314151617161920 2122232425262728293031323334353637363940

----------------------------------~-~---,~,~ -------------I-------------II--II------I-,o.o --------------------------------------m-8.9 -_-------_------_--_------------ -_--___ -8.4

7.7 7.5

--------------------------------------m-7.2 --------------------------~~--~~--~--~~-65

--s----m m---m -- - - -- -- 5.2 6.7 5.5

-----e-m m--m- - ---- -- - -- - s-----.1 -------------_---_--------B---m--- ------w--,5

------ -- - - - - -- -- --_-----s--m----- i.:

- ---- ---- --_----- -- ----a,5 ------ -- --- - ----- ---w-3,3

FIG. 3. As in Fig. 2 except that Hind111 digests of trypanosome DNA were hybridised to Lambda

In the previous work we have shown, using populations of trypanosome stocks col- lected from different areas, that four basic groupings could be used to define different trypanosome stocks: (1) T.b.gambiense stocks. (2) East African stocks which infect both man and animals and which appear to be indistinguishable from each other (clas- sically defined as T.b.rhodesiense, and T. b.brucei, respectively. (3) West African stocks which infect both man and animals and which appear to be indistinguishable from each other. By analogy we defined these as West African homologues of the East African T.b.rhodesienselT.b.brucei group of trypanosome. (4) T.b.rhodesiense stocks from Zambia. Thus, we now have a set of parameters with which to compare other stocks to assess their identity.

We have extended our analysis of try- panosome stocks, using this repetitive DNA system, to investigate stocks isolated from the major sleeping sickness foci in Zambia, Kenya, and Uganda.

Characterisation of Trypanosoma brucei rhodesiense Stocks

Luangwa Valley focus in Zambia. The Luangwa Valley in Zambia was considered to be the place of origin of T.b.rhodesiense sleeping sickness whence it spread north- ward over the ensuing years (Ormerod 1961). It lies in a tsetse fly belt, predomi- nantly inhabited by Glossina morsitans, and the river and its tributaries are the focal point for a number of villages, settlements, and homesteads. Typically patients who contracted sleeping sickness in this valley often showed mild symptoms (Foulkes 1970; Buyst 1974; Rickman 1974) and even some asymptomatic carriers were observed (Rickman 1974; Wurupa et al. 1984). A fur- ther characteristic of the epidemiology of sleeping sickness in this area is that there is a relatively high prevalence of disease in women and children (Rickman 1974; Buyst 1974; Dukes et al. 1984) suggesting that in- fection occurs within villages. In contrast a

1 * 3 4 5 6 7 8 9 10 11 12 13 14 15 16171819 20 2122 23 24 25 26 27 282930313233 34 3536 37 38 3940

I20 - 103 - -

- -- -- - ?eO ---- - --------- -- ------- _- ____ 95

-- -- - ---- ,": - ____------------ ---------------------81

_---- ------- --------------__- ____ 78

7.3 -- -- -- - - - - - 7.1

-- - - ____---------------- a------ -- ---- ;:

-------------------------------------~~ -------------------------------------L2

----------------------------------------4,5

----------------------------------------4,3

___------------------------------- ------,, 6

FIG. 4. As in Fig. 2 except that Hind111 digests of trypanosome DNA were hybridised to pBE2.

CHARACTERISATION OF T.b.rhodesiense STOCKS 435

1 2 3 4 5 6 7 8 9 10 11 121314 15 16 1718 19 20 2122232425 262728293031323334 35383738 39 40

--

--1111111-1111---1

-- -- --

---w - --

___- - ----- -

--- -

--- -

-----w--------s---

--- ----- -

--

--

--------------mm--

--1--1----1--

- -

s-e --- - --_

s-w - -

s--m

--m-------w__

----- -------_

- - B----w___

--- -----we--

---------------- -

-- __e------------

-- _----_-__-----

---__------__-----

---------_____

-m-m ---------

m-m- - --

m--m

-------- “,‘: -------- 3.5 --------a,*

- - - - 2.8 2.4

FIG. 5. As in Fig. 2 except that XhoI digest2 i of trypanosome DNA were hybl idised to Lambda 109.

---m----135 --------I23

t1.2 ,--,,,,,104

9.7 -- 8.9

-- -----8.7

:.i --------7.2 -- ----me,

- -----6.0 - 5.7 --------5.0

4.9

different epidemiological profile of T. b. rhodesiense sleeping sickness was ob- served in the more northerly foci in Kenya and Uganda. In these areas the disease pro- tile was very acute (MacKichan 1944) and a higher prevalence of disease was observed in Uganda, (Abaru 1985) and Kenya (Gib- son and Wellde 1985) than was found in the Luangwa Valley (Dukes et al. 1983, 1984; Wurupa et al. 1984). Also the prevalence was found to be much higher in adult males suggesting that the disease was contracted at work, outside the village environment (Abaru 1985). It has been presumed that as T.b.rhodesiense spread northward changes occurred in its level of virulence thus ex- plaining these epidemiological differences (Ormerod 1961).

In 1982, a sleeping sickness outbreak oc- curred in a small village, Kasyasya, in the North of the Luangwa Valley. This out- break was unusual because of the very high prevalence observed. From a population of 76 villagers, 15.8% were reported to have sleeping sickness (Dukes et al. 1983). By comparison, in a survey of 1093 people, in the Chibale area of Zambia, the prevalence was found to be only 0.64% (Dukes et al. 1983, 1984).

An important area of investigation into the epidemiology of sleeping sickness in the Luangwa Valley concerns the origins and types of trypanosome strains responsible for this disease. Two pertinent questions can be asked. First, what is the nature of the differences between the strains causing

disease in the Northern foci and those re- sponsible for the disease in Zambia? Sec- ondly, what factors are responsible for the high incidence of disease in Kasyasya Vil- lage?

To answer these types of questions, a number of techniques have been deployed to characterise trypanosome strains. Anal- ysis of isoenzyme variation has been the main method of analysis of trypanosome stocks. Using isoenzyme analysis, Gibson et al. (1980) examined a number of trypano- some stocks isolated from the Luangwa Valley in 1971-1974. The isoenzyme pat- terns observed in the human infective try- panosomes were quite distinct from other stocks isolated in the Northern foci in Kenya and Uganda. More recently, God- frey et al. (1990) have confirmed this gen- eral observation based on a larger sample of stocks. Isoenzyme analysis of stocks taken from the outbreak in Kasyasya showed that two very closely related zymodemes were present in the 12 isolates taken during that study (Dukes et al. 1983). This suggested that the spread of the disease was occurring by close man-fly-man transmission of a closely related pair of strains (Dukes et al. 1983).

In this work we have analysed the rela- tionships between isolates of trypanosomes by cluster analysis using repetitive DNA banding patterns. The first observation from the groups shown in the dendrogram is that the trypanosome stocks isolated in Zambia are quite distinct from those iso-

436 HIDE ET AL.

2209 TREU 881 TREU927 LUMP 258 TREU 1087 ousou YA

Group 1 Group 2

Grow 3 .-.-. hlos El, NIPA OURAM Gro”p 4 FOUNA ZENOU ELIANE 1871 2290

:::: 2222 2269

2274 BUP2S40 Group 5 GUP 2546 GUP 2560 IXIP 2560

r L

AMA

q!!ij

NITR4OI12 TAEU la96 TREU 1096 TREU 1397 TREU 1395 TREU 1399

Group 6

Group 7

,

40 60 70

Similarity (%)

FIG. 6. A dendrogram showing the relationships of 80 trypanosome stocks examined in this and a previous study (Hide et al. 1990) as determined by comparisons of banding patterns. The degree of similarity of groups is indicated in the scale (measured as a percentage of complete identity) and each group formed by the cluster analysis is indicated (see results).

CHARACTERISATION OF T.b.rhodesiense STOCKS 437

lated from the northern foci in Kenya and Uganda. They are also clearly not T.b. gambiense stocks as they do not fall into group 4. The differences between the stocks isolated in Zambia and those iso- lated in the Kenya/Uganda area are as great as the differences between T.b.gambiense and T.b.rhodesiense. Thus, it seems un- likely that the Kenyan and Ugandan foci were established by a northerly spread of strains from Zambia as previously sug- gested (Ormerod 1961). The origins of the human infective stocks in these foci are, as yet, unclear but perhaps they emerged by independent events from the background animal and fly populations.

The stocks isolated from the village of Kasyasya can be seen, by examination of the dendrogram in Fig. 6, to group along- side stocks isolated from another area of Zambia (Chibale) . Therefore, there is noth- ing strikingly different about the stocks re- sponsible for the Kasyasya epidemic and those found elsewhere in the Luangwa Val- ley. The reason for the high incidence in Kasyasya would appear to be due to a pe- culiarity in the epidemiological relation- ships of man, vector, and reservoirs rather than the appearance of a new strain. On the other hand, the stocks isolated from Kas- yasya were all very similar if not identical suggesting that a single, or very few, strains were responsible for the disease. The most probable reason for this epidemic therefore is that the strain (or strains) were transmit- ted around the village by close man- fly-man contact as proposed previously by Dukes et al. (1983). One stock, 2208, had banding patterns which were quite different from any other stock examined. As this stock was obtained from the same house- hold as other isolates (for example GUP2590) and previously isoenzyme stud- ies had shown 2208 to have an identical isozyme pattern to the other stocks from this household (Dukes et al. 1983), we con- clude that this inconsistancy is due to the stock being wrongly coded in the time be-

tween the analysis of Dukes et al. (1983) and this study.

One of the Kasyasya stocks, GUP 2560, was isolated from a sentinel goat in Kas- yasya. This has a pattern identical to three other stocks isolated from patients in the village, again suggesting the spread of a sin- gle trypanosome strain throughout this vil- lage.

Northern foci in Kenya and Uganda. The first evidence of T.b.rhodesiense sleeping sickness in the Kenya/Uganda region came in 1940 when an epidemic occurred in the Busoga region of Uganda (MacKichan 1944; Ormerod 1961). The epidemic then spread across the Kenyan border through the Samia and Nyanza regions. These areas have remained sleeping sickness foci ever since.

In the Busoga region of Uganda, there was an increase in the prevalence of sleep- ing sickness from 1971 onward (Abaru 1985). This epidemic peaked between the years of 19761982 and the trypanosomes responsible for it have been the subject of other studies using isoenzyme variation (Gibson et al. 1980; Gibson and Gashumba 1983; Godfrey et al. 1990). These studies showed that the cases of sleeping sickness present prior to 1976 could be accounted for by the spread of a single trypanosome strain (Gibson et al. 1980). However, anal- ysis of more recent stocks has shown that at least six trypanosome strains were circulat- ing (Gibson and Gashumba 1983). It was postulated that some of these stocks arose by recombination between the “original” Ugandan strain and incoming strains from Ethiopia or Zambia while others may have been derived from the Nyanza foci in Kenya (Gibson and Gashumba 1983).

The Central and South Nyanza regions are two of the major sleeping sickness foci in Kenya. Isoenzyme analysis of stocks from the Central Nyanza region suggested that the trypanosome strains responsible for that epidemic had come from Uganda while the stocks isolated in South Nyanza

438 HIDE ET AL.

clearly had different origins and may have come from Tanzania in the South.

In this study we have included a number of stocks isolated from the Busoga area of Uganda and the Central Nyanza region of Kenya. All of these stocks fall into groups 7, 8, and 9. The stocks which fell into groups 8 and 9 were isolated, recently, in the Iyolwa subcounty of South East Uganda from cattle and pigs and were all found to be human serum sensitive (Maud- lin er al., in preparation); that is, these stocks were T.b.brucei animal infective stocks. Another set of stocks, also recently isolated in Iyolwa, fall into the large group 7 alongside other known T.b.rhodesiense stocks. These stocks were isolated from man and cattle and were all shown to be human serum resistant (Maudlin and Wel- bum, in preparation). They had very simi- lar banding patterns to stocks previously isolated from humans in Busoga in 1958, 1981, and 1982. Thus, the cases of sleeping sickness in this area were probably caused by the same trypanosome stocks which have been circulating since 1958. Stocks isolated from cattle and pigs at the same time and place had identical banding pat- terns thus confirming that domestic live- stock act as a reservoir for sleeping sick- ness in this area.

In addition to the Iyolwa T. b.rhodesiense stocks, group 7 also contains a large num- ber of stocks whose banding patterns are relatively homogeneous. The homogeneity of this group suggests that they may have common ancestral origins. A collection of stocks from the Central Nyanza focus were found to fall within this group. These stocks were isolated between 1961 and 1977 and representative stocks from man, cattle, and tsetse were examined. The human infective stocks from this group have banding pat- terns which are at the 80% level (or greater) similar to the human infective stocks from Uganda, suggesting that they may have common origins. In some cases evidence can be seen for the existence of the same strain in the two areas. For example, UTRO

4, GUP 1301, and GUP 1052 have identical patterns and therefore the strain was present in the human population in Kenya in 1961 and Uganda in 1981 and was also found in the cattle population in the Ugandan outbreak.

In addition to the Kenyan and Ugandan human infective isolates, this group also con- tained the human-infective stock, STIB 386, which was isolated in West Africa but previ- ously shown to have banding patterns indic- ative of East African origins (Hide 1988; Hide et al. 1990). Thus, the grouping of this stock remains stable in this analysis despite the ad- dition of more stocks to the analysis.

General epidemiology and evolution of T.b.rhodesiense in south and east Africa. In this analysis, we have compared T.brucei stocks from Zambia and Kenya/Uganda. The differences observed between these groups of stocks are as great as those between T.b.gambiense and T. b.brucei and do not wholly support the hy- pothesis that T.b.rhodesiense sleeping sick- ness arose in the Luangwa Valley and spread northward to the Kenyan/Ugandan foci (Ormerod 1961). This is further sup- ported by the fact that identical strains (as assessed by the banding patterns studied here and isoenzyme patterns (Gibson and Wellde 1985)) were present in the Kenyan/Ugandan foci between the years of 1961 and 1982. The implication of this is that the composition of strains circulating within a given focus and moving between foci (e.g. Busoga to Central Nyanza) is rel- atively stable. We might have expected some degree of identity between the Zam- bian and Kenyan/Ugandan stocks had there been common origins. The origins of this divergence are unknown; it may be that try- panosome strains acquire human serum re- sistance (i.e., human infectivity) as an in- dependent event arising from the back- ground tsetse or animal populations followed by clonal propagation (Tibayrenc 1990) enhanced by appropriate host-vector cycles and finally the acquisition of some diversity by mating and genetic exchange (Tait 1980; Gibson et al. 1980).

CHARACTERISATION OF T. b.rhodesiense STOCKS 439

ACKNOWLEDGMENTS We thank the following organisations for financial

support: The Wellcome Trust for continued support (G.H., N.B., S.W., J.D.B., A.T.); A grant from the World Health Organisation (J.D.B., A.T.); Overseas Development Administration of the U.K. Government and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) (IM). J.D.B. is a Wellcome Senior Lecturer. We also thank Alan May for the photography and Afshan Fair- ley for the manuscript. We especially thank Richard Cibulskis (University of Liverpool) for kindly provid- ing computer programs, which have greatly assisted the cluster analysis, and Peter Dukes (University of Bristol) for helpful discussion.

GODFREY, D. G., BAKER, R. D., RICKMAN, L. R., AND MEHLITZ, D. 1990. The distribution, relation- ships, and identification of enzymic variants within the subgenus. Trypanozoon Advances in Parasitol- ogy 29, l-74.

HIDE, G. 1988. Variation in repetitive DNA in African trypanosomes. PhD. thesis, University of Edin- burgh.

HIDE, G., CATTAND, P., LE RAY D., BARRY, J. D., AND TAIT, A. 1990. The identification of Trypano- soma brucei subspecies using repetitive DNA se- quences. Molecular and Biochemical Parasitology 39, 213-226.

MACKICHAN, I. W. 1944. Rhodesian sleeping sickness in eastern Uganda. Transactions of the Royal Soci- ety of Tropical Medicine and Hygiene 38, 49.

REFERENCES OR~E~OD, W. E. 1961. The epidermic spread of Rho-

AIJARU, D. E. 1985. Sleeping sickness in Busoga, desian sleeping sickness 1908-1960. Transactions of

Uganda, 1976-1983. Tropical Medicine and Parasi- the Royal Society of Tropical Medicine and Hygiene

tology 36, 72-76. 55, 525-538.

BARRY, J. D., CROWE, J. S., AND VICKERMAN, K. RICKMAN, L. R. 1974. Investigations into an outbreak

1983. Instability of the Trypanosoma rhodesiense of human trypanosomiasis in the lower Luangwa

metacyclic variable antigen repertoire. Nature 306, Valley, Eastern Province, Zambia. East African

699-70 1. Medical Journal 51, 467-487.

BUYST, H. 1974. The epidemiology, clinical features, SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T.

treatment and history of sleeping sickness on the 1989. Molecular Cloning: A laboratory manual. Sec-

northern edge of the Luangwa Fly belt. Medical ond ed. Cold Spring Harbor Laboratory Press, Cold

Journal of Zambia 8, 2-12. Spring Harbor, NY.

DUKES, P., SCOTT, C. M., RICKMAN, L. R., AND Wu- STEPHENS, J. W. W., AND FANTHAM, H. B. 1910. On

PARA, F. 1983. Sleeping sickness in the Luangwa the peculiar morphology of a trypanosome from a

Valley of Zambia. A preliminary report of the 1982 case of sleeping sickness and the possibility of its

outbreak at Kasyasya Village. Bulletin de la Societe being a new species (T.rhodesiense). Proceedings of

de Pathologie Exotique 76, 605-613. the Royal Society, Series B 83, 28.

DUKES, P., RICKMAN, L. R., KILLICK-KENDRICK, R., TAIT, A. 1980. Evidence for diploidy and mating in

KAKOMA, I., WURAPA, F. K., DE RAADT, P., AND trypanosomes. Nature 287, 536-538.

MORROW, R. 1984. A field comparison of seven di- TAIT, A., BABIKER, E. A., AND LE RAY, D. 1984. En-

agnostic techniques for human trypanosomiasis in zyme variation in T.brucei spp. I. Evidence for the

the Luangwa Valley, Zambia. Tropenmedezin und subspeciation of T.b.gambiense. Parasitology 89, 311-326.

Parasitology 35, 141-147. FOULKES, J. 1970. Human trypanosomiasis in Zambia.

TAIT, A., BARRY, J. D., WINK, R., SANDERSON, A.,

Medical Journal of Zambia 4, 167-177. AND CROWE, J. S. 1985. Enzyme variation in T.bru-

GIBSON, W. C., AND GASHUMBA, J. K. 1983. Isoen- cei ssp. II. Evidence for T.b.rhodesiense being a set

zyme characterisation of some Trypanozoon stocks of variants of T.b.brucei. Parasitology 90, 89-100.

from a recent trypanosomiasis epidemic in Uganda. TIBAYRENC, M., KJELLBERG, F., AND AYALA, F. J.

Transactions of the Royal Society of Tropical Med- 1990. A clonal theory of parasitic protozoa: The

icine and Hygiene 77, 114-l 18. population structures of Entamoeba, Giardia,

GIBSON, W. C., AND WELLDE, B. T. 1985. Character- Leishmania, Naegleria, Plasmodium, Trichomonas

isation of Trypanozoon stocks from the South and Trypanosoma and their medical and taxonomi-

Nyanza sleeping sickness focus in western Kenya. cal consequences. Proceedings of the National

Transactions of the Royal Society of Tropical Med- Academy of Sciences, USA 87, 2414-2418.

icine and Hygene 79, 671-676. WURUPA, F., DUKES, P., NJELESANI, E. K., AND

GIBSON, W. C., MARSHALL, T. F. DE C., AND GOD- BOATIN, B. 1984. A “healthy carrier” of Trypano-

FREY, D. G. 1980. Numerical analysis of enzyme soma rhodesiense: A case report. Transactions of

polymorphism. A new approach to the epidemiology the Royal So&sty of Tropical Medicine and Hygiene

and taxonomy of trypanosomes of the subgenus 78, 349-350.

Trypanozoon. Advances in Parasitology 18, 175- Received 22 August 1990; accepted with revision 16 245. November 1990