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 followed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Parasitol. 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 1991kademi~ press,Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma brucei brucei; T.b.rhodesiense; T.b.gambiense; Kinetoplastida; Protozoa; Repetitive DNA; Ribosomal RNA genes; Molecular epidemiology; Strain characterisation; Epidemiology; Zambia; Kenya; Uganda; Cluster analysis; Southern blotting; Deoxyribonucleic acid (DNA); Ribonucleic acid (RNA); Sodium dodecyl sulphate (SDS).
INTRODUCTION
In a previous study, we developed a system 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 trypanosome stocks, were used to characterise the
degree of relatedness between the stocks using a cluster analysis technique. This approach has been used here as a tool for examining the relationships and epidemiology of Trypanosoma brucei rhodesiense stocks from Kenya, Uganda, and Zambia. The first recorded cases of sleeping sickness caused by Ttypanosoma brucei rhodesiense 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
with another 50 stocks taken from a previous analysis (Hide et al. 1990). The probes used include Lambda 104, a probe which represents the ribosomal coding region, Lambda 109, which represents the nontranscribed spacer region, and pBE2 an uncharacterised 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 number of such blots is presented in Figs. 2-5. MATERIALS ANDMETHODS The four sets of data obtained from the Trypanosome stocks. The trypanosome stocks ex- Southern analysis are as follows: Hind111 amined in this study are described in Table I and in digests of trypanosome DNA probed with Hide et al. (1990). Lambda 104 (Fig. 2), Lambda 109 (Fig. 3), Isolation of trypanosome DNA, plasmid, and phage and pBE2 (Fig. 4) and XhoI digested tryDNA probes. Isolation of DNA from trypanosomes and DNA probes (lambda 104, lambda 109, and pBE2) panosome DNA probed with Lambda 109 was carried out as previously described (Hide 1988; (Fig. 5). Hide et al. 1990). Cluster analysis was applied to the data Southern blotting. Southern blotting was carried out on banding patterns obtained from the as previously described (Hide et al. 1990) except that Southern blotting to determine the relatedDNA was transferred on to Nylon filters (Amersham Hybond-N), instead of nitrocellulose, and the DNA ness of each stock based on the similarity was fixed to the filter by cross-linking by exposure to and differences between banding patterns. long-wave ultraviolet light for 7 min. The filter was The results are shown in the dendrogram prehybridised for 30 min in 0.5 M phosphate buffer, pH 7.2, 7% SDS at 65°C and hybridised in IO’ cpm of illustrated in Fig. 6. From the dendrogram it can be seen that nine distinct groups of the appropriate DNA probe overnight at 65°C in 0.5 M phosphate buffer, pH 7.2, 7% SDS and washed twice T.brucei stocks are formed. A summary of for 15 min each wash in 0.1% SDS, 2 x SSC (Sarn- the composition of each group of stocks is brook et al. 1989) at 65°C. These modifications of the given below: Southern hybridisation method gave identical results Group 1. This group contains a single huto the previously described method (Hide et al. 1990). Cluster analysis. Cluster analysis was carried out as man-infective stock, 2208, which was isopreviously described (Hide et nl. 1990) except that lated from the village of Kasyasya in Zamcomputer programs were used to construct the simi- bia. larity matrices (BIGSIM) and dendrograms (4M). Group 2. This group comprises three BIGSIM was a modification of the program SIM (writstocks isolated from tsetse in the Kiboko ten by R. E. Cibulskis, University of Liverpool) to region of Kenya. handle larger data sets. Group 3. Two stocks from West Africa, one of which was isolated from man and the RESULTS other from cattle, are contained within this DNA sequences representing the riboso- group. These stocks represent the West Afma1 RNA genes of T.brucei were used to rican “rhodesiense’‘-like trypanosomes. Group 4. This group contains the T.b. probe Southern blots of trypanosome DNA from a number of T.b.rhodesiense stocks gambiense stocks. Group 5. This group comprises, excluisolated from the Zambian, Kenyan, and Ugandan foci. The 40 stocks used in this sively, human-infective stocks isolated analysis are listed in Table I and the results from Zambia. obtained from the analysis were combined Group 6. The stocks in this group are 1944; Foulkes 1970; Buyst 1974; Rickman 1974) of T.b.rhodesiense-associated sleeping sickness between the major disease foci in Kenya/Uganda and those in Zambia. We have set out to investigate the epidemiological relationships between T.b.rhodesiense stocks isolated from these major foci in Kenya, Uganda, and Zambia using the repetitive DNA system.
432
HIDE
Trypano~omn Stock
ET AL.
TABLE I brucei stocks used in this analysis
Other designation
Host
Date
Location
Kenyan stocks GUP793 GUPlo42 GUPlo43 GUPlOSl GUP1052 GUP2077 GUP2078 EATR0156 EATR0795 GUP2498
GPAL/KE/6lIEATROl8 MHOM/KE/6l/EATR094 MHOM/KE/6l/EATR095 MHOM/KE/6l/EATRO% MHOM/KE/6l/EATRO97 MHOM/KE/6l/EATR0106 MHOM/KEMl/EATROll6 MHOM/KE/6l/EATROl56 MBOI/KE/ti/EATR0795 MHOM/KE/77iEATR02340
Tsetse MElll Man Man Man Man Mall Man Cattle Man
1%1 1%1 1%1 1961 1961 1%1 1961 1961 1964 1977
Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Alego, Samia,
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 GPAL/UG/6O/EATR03 MHOM/UGI8lIUTRO2 MHOM!UG/8l/UTR03 MHOM/UG/8l/UTR04 MBOYUG/88/IYOLWA96 MHOM/UG/BS/UGANDA MHOMIUGI88IUGANDA MBOYUG/88/IYOLWAl25 MBOIfLJG/SS/MELA27 MBOI/UG/88/IYOLWAl02 MHOM/UG/88/UGANDA MBOI/UG/88/MELA71 MBOYUG/88/PAPOL42 MSUS/UG/88/MELA PIG MBOYUG/88/MELA2 MBOI/UG/88/POYEMll MBOVUG/88/IYOLWAl53 MHOM/UG/88/UGANDA MHOM/UG/88/UGANDA MHOMIUGI88IUGANDA MHOM/UG/88/UGANDA
Man Tsetse Man Man Man Cattle Man Man Cattle Cattle Cattle Man Cattle Cattle m Cattle Cattle Cattle Man Man Man Man
1959 1960 1981 1981 1982 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988
Busoga, Busoga, Busoga, Busoga, Busoga, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa,
Zambian stocks 2194 2208 2210 2218 2222 2269 2274 2290
MHOMIZM/8UZl94 MHOM/ZM/82/Z208 MHOM/ZM/82/Z210 MHOM/ZM/82/Z218 MHOMlZMl82JZ222 MCAPlZMl83iZ269 MHOM/ZM/82/Z274 MHOM/ZM/8l/Z%
Man Man Man Man MkUl Goat Man Man
1982 1982 1982 1982 1982 1983 1982 1981
Chibale, Zambia Kasyasya, Zambia Kasyasya, Zambia Chibale, Zambia Kasyasya, Zambia Kasyasya, Zambia Kasyasya, Zambia Chibale, Zambia
A F
H
B C E G
from West Africa and were isolated both from humans and animals. As with group 3, these stocks represent West African “rhodesiense’‘-like trypanosomes. Group 7. This group contains a large
Kenya Kenya Kenya Kenya Kenya Kenya Kenya Kenya Kenya Kenya Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda
number of stocks isolated from animals (cattle and sheep), tsetse, and man from Kenya and Uganda. Group 8. The stocks contained within this group are all isolated from cattle in
OF T. b.rhodesiense
CHARACTERISATION
433
STOCKS
tem, based on similarities and differences in banding patterns generated by repetitive DNA probes, to characterise the relationships between trypanosome stocks (Hide et al. 1990). The main advantage of this system is that it is able to establish levels of identity and difference between trypanosome stocks, based on variation in multiple loci, without dependence on other biological information available on the stock. The sensitivity of this approach is enhanced by the use of a large number of characters FIG. 1. A typical Southern blot showing banding (bands) such that high degrees of similarity patterns produced when Hind111 digested trypanosome DNA was probed with pBE2. (A) A comparison in banding patterns reflect close genetic of Kenyan T.brucei stocks, TREU 869 (lane l), identity. It is not possible to place exact EATRO 156 (lane 3), GUP 1043 (lane 4), and stocks probabalistic values on the confidence with isolated from Zambia, Z 290 (lane 2) and Z 269 (lane 5). which genetic identity and difference can (B) A comparison of trypanosome stocks from Uganda. UG 14, 15, 16, and 17 (lanes 3-6, respec- be established, using this approach, as no tively) were isolated from man, and UG 12 and 13 information is available on rates of change (lanes 1 and 2, respectively) were human serum sen- of these banding patterns in trypanosome sitive trypanosomes isolated from animals. Sizes of field isolates. However, the sensitivity of some of the bands are given in kilobases for reference this approach was assessed by a comparipurposes. son, carried out blind, with isoenzyme and biological characteristics obtained from Uganda and are human serum sensitive other work (Maudlin et al., in preparation), (i.e., not human infective). using a collection of trypanosome stocks Group 9. The stocks contained within from Uganda (UGl-17). Complete agreethis group are isolated from pigs and cattle ment between these different approaches in Uganda and are human serum sensitive. was observed. This system is, therefore, capable of answering a number of imporDISCUSSION tant epidemiological questions which may In a previous study, we have used a sys- not be answerable using other approaches. B
A
123456
12345
1 2 3 4 5 6 7 6 ----------------------------------------,a ----------------I-----------------------~~ _____m-e--v-------------------,,,,,1,,-11------,,-,,,,,,,,,1,,,,,,,,,,~~ ___________--_-------------
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910111213141516171819
202122232425262726293031323334353637363940 -
---------------s--w
-_-
-
-
---
-
-
-
-w-m-
-
-
-
-w-m-
-
-
-
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-
-
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-
-
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--
----j; 35
-----------e-----35 -
--
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----80
-----
-
--
-
-
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 -_-------_------_--_------------
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--------------------------------------m-7.2 --------------------------~~--~~--~--~~-65 --s----m
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--
-----
5.2 6.7 5.5
i.:
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 collected 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 (classically 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 trypanosome stocks, using this repetitive DNA system, to investigate stocks isolated from the major sleeping sickness foci in Zambia, Kenya, and Uganda. 1
*
3
4 5
6
7 8
9
-
14
15 16171819
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 northward over the ensuing years (Ormerod 1961). It lies in a tsetse fly belt, predominantly 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 further 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 infection occurs within villages. In contrast a
20 2122
23 24 25 26 27 282930313233
34 3536
37 38 3940 I20 103
--
-----
10 11 12 13
of Trypanosoma brucei rhodesiense Stocks
Characterisation
---------
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,":
---------------------81 --------------__-
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78 7.3 7.1
----
;:
--
-------------------------------------~~ -------------------------------------L2 ----------------------------------------4,5 ----------------------------------------4,3 ___-------------------------------
------,,
FIG. 4. As in Fig. 2 except that Hind111 digests of trypanosome DNA were hybridised to pBE2.
6
OF T.b.rhodesiense
CHARACTERISATION 1 2
3
4
5
6
7 8
9 10 11 121314
15 16 1718
19 20 2122232425
435
STOCKS
262728293031323334
35383738
39 40
---m----135 --------I23 t1.2
--
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--
9.7 8.9
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---
-
s-e
---
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-
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--------7.2 ---------5.0
---------
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--m-------w__ -----
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-----8.7
s--m
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--
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----me, -----6.0 5.7 4.9
--------------------__------__-----
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---------_____ -m-m m-mm--m
-
--
“,‘: 3.5 -
-
-
-
2.8 2.4
FIG. 5. As in Fig. 2 except that XhoI digest2i of trypanosome DNA were hybl idised to Lambda 109.
different epidemiological profile of T. b. rhodesiense sleeping sickness was observed in the more northerly foci in Kenya and Uganda. In these areas the disease protile was very acute (MacKichan 1944) and a higher prevalence of disease was observed in Uganda, (Abaru 1985) and Kenya (Gibson 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 explaining these epidemiological differences (Ormerod 1961). In 1982, a sleeping sickness outbreak occurred in a small village, Kasyasya, in the North of the Luangwa Valley. This outbreak 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 responsible for the disease in Zambia? Secondly, what factors are responsible for the high incidence of disease in Kasyasya Village? To answer these types of questions, a number of techniques have been deployed to characterise trypanosome strains. Analysis of isoenzyme variation has been the main method of analysis of trypanosome stocks. Using isoenzyme analysis, Gibson et al. (1980) examined a number of trypanosome stocks isolated from the Luangwa Valley in 1971-1974. The isoenzyme patterns observed in the human infective trypanosomes were quite distinct from other stocks isolated in the Northern foci in Kenya and Uganda. More recently, Godfrey et al. (1990) have confirmed this general 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 relationships 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
.-.-. hlos El,
NIPA OURAM FOUNA ZENOU ELIANE 1871 2290
Group 1 Group 2 Grow 3
Gro”p
4
Group
5
:::: 2222 2269 2274 BUP2S40 GUP 2546 GUP 2560 IXIP 2560
r
NITR4OI12 TAEU la96 TREU 1096 TREU 1397 TREU 1395 TREU 1399
L
Group 6
AMA
q!!ij
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
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 isolated in the Kenya/Uganda area are as great as the differences between T.b.gambiense and T.b.rhodesiense. Thus, it seems unlikely that the Kenyan and Ugandan foci were established by a northerly spread of strains from Zambia as previously suggested (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 alongside stocks isolated from another area of Zambia (Chibale) . Therefore, there is nothing strikingly different about the stocks responsible for the Kasyasya epidemic and those found elsewhere in the Luangwa Valley. The reason for the high incidence in Kasyasya would appear to be due to a peculiarity in the epidemiological relationships of man, vector, and reservoirs rather than the appearance of a new strain. On the other hand, the stocks isolated from Kasyasya 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 transmitted around the village by close manfly-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 household as other isolates (for example GUP2590) and previously isoenzyme studies had shown 2208 to have an identical isozyme pattern to the other stocks from this household (Dukes et al. 1983), we conclude that this inconsistancy is due to the stock being wrongly coded in the time be-
T.b.rhodesiense STOCKS
437
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 Kasyasya. This has a pattern identical to three other stocks isolated from patients in the village, again suggesting the spread of a single trypanosome strain throughout this village. 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 sleeping 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, analysis of more recent stocks has shown that at least six trypanosome strains were circulating (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
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 (Maudlin 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 Welbum, in preparation). They had very similar 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 patterns thus confirming that domestic livestock act as a reservoir for sleeping sickness in this area. In addition to the Iyolwa T. b.rhodesiense stocks, group 7 also contains a large number 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 patterns 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
ET AL.
4, GUP 1301, and GUP 1052 have identical patterns and therefore the strain was present in the human population in Kenya in 1961and 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 contained the human-infective stock, STIB 386, which was isolated in West Africa but previously shown to have banding patterns indicative of East African origins (Hide 1988;Hide et al. 1990). Thus, the grouping of this stock remains stable in this analysis despite the addition 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 hypothesis that T.b.rhodesiense sleeping sickness arose in the Luangwa Valley and spread northward to the Kenyan/Ugandan foci (Ormerod 1961). This is further supported 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 relatively stable. We might have expected some degree of identity between the Zambian and Kenyan/Ugandan stocks had there been common origins. The origins of this divergence are unknown; it may be that trypanosome strains acquire human serum resistance (i.e., human infectivity) as an independent event arising from the background 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 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 Fairley for the manuscript. We especially thank Richard Cibulskis (University of Liverpool) for kindly providing computer programs, which have greatly assisted the cluster analysis, and Peter Dukes (University of Bristol) for helpful discussion.
REFERENCES AIJARU, D. E. 1985. Sleeping sickness in Busoga, Uganda, 1976-1983. Tropical Medicine and Parasitology 36, 72-76.
BARRY, J. D., CROWE, J. S., AND VICKERMAN, K. 1983. Instability of the Trypanosoma rhodesiense metacyclic variable antigen repertoire. Nature 306, 699-70 1. BUYST, H. 1974. The epidemiology, clinical features, treatment and history of sleeping sickness on the northern edge of the Luangwa Fly belt. Medical Journal
of Zambia 8, 2-12.
DUKES, P., SCOTT,C. M., RICKMAN, L. R., AND WuPARA, F. 1983. Sleeping sickness in the Luangwa Valley of Zambia. A preliminary report of the 1982 outbreak at Kasyasya Village. Bulletin de la Societe de Pathologie Exotique 76, 605-613. DUKES, P., RICKMAN, L. R., KILLICK-KENDRICK, R., KAKOMA, I., WURAPA, F. K., DE RAADT, P., AND MORROW,R. 1984. A field comparison of seven diagnostic techniques for human trypanosomiasis in the Luangwa Valley, Zambia. Tropenmedezin und Parasitology 35, 141-147. FOULKES,J. 1970. Human trypanosomiasis in Zambia. Medical
Journal
of Zambia 4, 167-177.
GIBSON, W. C., AND GASHUMBA, J. K. 1983. Isoenzyme characterisation of some Trypanozoon stocks from a recent trypanosomiasis epidemic in Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 114-l 18.
GIBSON,W. C., AND WELLDE, B. T. 1985. Characterisation of Trypanozoon stocks from the South Nyanza sleeping sickness focus in western Kenya. Transactions of the Royal Society of Tropical Medicine and Hygene 79, 671-676.
GIBSON, W. C., MARSHALL, T. F. DE C., AND GODFREY, D. G. 1980. Numerical analysis of enzyme polymorphism. A new approach to the epidemiology and taxonomy of trypanosomes of the subgenus Trypanozoon. Advances in Parasitology 18, 175245.
439
STOCKS
GODFREY, D. G., BAKER, R. D., RICKMAN, L. R., AND MEHLITZ, D. 1990. The distribution, relationships, and identification of enzymic variants within the subgenus. Trypanozoon Advances in Parasitology 29, l-74. HIDE, G. 1988. Variation in repetitive DNA in African trypanosomes. PhD. thesis, University of Edinburgh. HIDE, G., CATTAND, P., LE RAY D., BARRY, J. D., AND TAIT, A. 1990. The identification of Trypanosoma brucei subspecies using repetitive DNA sequences. Molecular and Biochemical Parasitology 39, 213-226. MACKICHAN, I. W. 1944.Rhodesian sleeping sickness in eastern Uganda. Transactions of the Royal Society of Tropical
Medicine
and Hygiene
38, 49.
OR~E~OD, W. E. 1961. The epidermic spread of Rhodesian sleeping sickness 1908-1960. Transactions of the Royal Society of Tropical Medicine
and Hygiene
55, 525-538. RICKMAN, L. R. 1974. Investigations into an outbreak of human trypanosomiasis in the lower Luangwa Valley, Eastern Province, Zambia. East African Medical Journal 51, 467-487. SAMBROOK,J., FRITSCH, E. F., AND MANIATIS, T. 1989. Molecular Cloning: A laboratory manual. Second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. STEPHENS,J. W. W., AND FANTHAM, H. B. 1910. On the peculiar morphology of a trypanosome from a case of sleeping sickness and the possibility of its being a new species (T.rhodesiense). Proceedings of the Royal Society, Series B 83, 28.
TAIT, A. 1980. Evidence for diploidy and mating in trypanosomes. Nature 287, 536-538. TAIT, A., BABIKER, E. A., AND LE RAY, D. 1984. Enzyme variation in T.brucei spp. I. Evidence for the subspeciation of T.b.gambiense. Parasitology 89, 311-326. TAIT, A., BARRY, J. D., WINK, R., SANDERSON,A., AND CROWE,J. S. 1985. Enzyme variation in T.brucei ssp. II. Evidence for T.b.rhodesiense being a set of variants of T.b.brucei. Parasitology 90, 89-100. TIBAYRENC, M., KJELLBERG,F., AND AYALA, F. J. 1990. A clonal theory of parasitic protozoa: The population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas and Trypanosoma and their medical and taxonomical consequences. Proceedings of the National Academy of Sciences, USA 87, 2414-2418.
WURUPA, F., DUKES, P., NJELESANI, E. K., AND BOATIN, B. 1984. A “healthy carrier” of Trypanosoma rhodesiense: A case report. Transactions of the Royal So&sty of Tropical Medicine
and Hygiene
78, 349-350. Received 22 August 1990; accepted with revision 16 November 1990
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 followed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Parasitol. 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 1991kademi~ press,Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Trypanosoma brucei brucei; T.b.rhodesiense; T.b.gambiense; Kinetoplastida; Protozoa; Repetitive DNA; Ribosomal RNA genes; Molecular epidemiology; Strain characterisation; Epidemiology; Zambia; Kenya; Uganda; Cluster analysis; Southern blotting; Deoxyribonucleic acid (DNA); Ribonucleic acid (RNA); Sodium dodecyl sulphate (SDS).
INTRODUCTION
In a previous study, we developed a system 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 trypanosome stocks, were used to characterise the
degree of relatedness between the stocks using a cluster analysis technique. This approach has been used here as a tool for examining the relationships and epidemiology of Trypanosoma brucei rhodesiense stocks from Kenya, Uganda, and Zambia. The first recorded cases of sleeping sickness caused by Ttypanosoma brucei rhodesiense 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
with another 50 stocks taken from a previous analysis (Hide et al. 1990). The probes used include Lambda 104, a probe which represents the ribosomal coding region, Lambda 109, which represents the nontranscribed spacer region, and pBE2 an uncharacterised 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 number of such blots is presented in Figs. 2-5. MATERIALS ANDMETHODS The four sets of data obtained from the Trypanosome stocks. The trypanosome stocks ex- Southern analysis are as follows: Hind111 amined in this study are described in Table I and in digests of trypanosome DNA probed with Hide et al. (1990). Lambda 104 (Fig. 2), Lambda 109 (Fig. 3), Isolation of trypanosome DNA, plasmid, and phage and pBE2 (Fig. 4) and XhoI digested tryDNA probes. Isolation of DNA from trypanosomes and DNA probes (lambda 104, lambda 109, and pBE2) panosome DNA probed with Lambda 109 was carried out as previously described (Hide 1988; (Fig. 5). Hide et al. 1990). Cluster analysis was applied to the data Southern blotting. Southern blotting was carried out on banding patterns obtained from the as previously described (Hide et al. 1990) except that Southern blotting to determine the relatedDNA was transferred on to Nylon filters (Amersham Hybond-N), instead of nitrocellulose, and the DNA ness of each stock based on the similarity was fixed to the filter by cross-linking by exposure to and differences between banding patterns. long-wave ultraviolet light for 7 min. The filter was The results are shown in the dendrogram prehybridised for 30 min in 0.5 M phosphate buffer, pH 7.2, 7% SDS at 65°C and hybridised in IO’ cpm of illustrated in Fig. 6. From the dendrogram it can be seen that nine distinct groups of the appropriate DNA probe overnight at 65°C in 0.5 M phosphate buffer, pH 7.2, 7% SDS and washed twice T.brucei stocks are formed. A summary of for 15 min each wash in 0.1% SDS, 2 x SSC (Sarn- the composition of each group of stocks is brook et al. 1989) at 65°C. These modifications of the given below: Southern hybridisation method gave identical results Group 1. This group contains a single huto the previously described method (Hide et al. 1990). Cluster analysis. Cluster analysis was carried out as man-infective stock, 2208, which was isopreviously described (Hide et nl. 1990) except that lated from the village of Kasyasya in Zamcomputer programs were used to construct the simi- bia. larity matrices (BIGSIM) and dendrograms (4M). Group 2. This group comprises three BIGSIM was a modification of the program SIM (writstocks isolated from tsetse in the Kiboko ten by R. E. Cibulskis, University of Liverpool) to region of Kenya. handle larger data sets. Group 3. Two stocks from West Africa, one of which was isolated from man and the RESULTS other from cattle, are contained within this DNA sequences representing the riboso- group. These stocks represent the West Afma1 RNA genes of T.brucei were used to rican “rhodesiense’‘-like trypanosomes. Group 4. This group contains the T.b. probe Southern blots of trypanosome DNA from a number of T.b.rhodesiense stocks gambiense stocks. Group 5. This group comprises, excluisolated from the Zambian, Kenyan, and Ugandan foci. The 40 stocks used in this sively, human-infective stocks isolated analysis are listed in Table I and the results from Zambia. obtained from the analysis were combined Group 6. The stocks in this group are 1944; Foulkes 1970; Buyst 1974; Rickman 1974) of T.b.rhodesiense-associated sleeping sickness between the major disease foci in Kenya/Uganda and those in Zambia. We have set out to investigate the epidemiological relationships between T.b.rhodesiense stocks isolated from these major foci in Kenya, Uganda, and Zambia using the repetitive DNA system.
432
HIDE
Trypano~omn Stock
ET AL.
TABLE I brucei stocks used in this analysis
Other designation
Host
Date
Location
Kenyan stocks GUP793 GUPlo42 GUPlo43 GUPlOSl GUP1052 GUP2077 GUP2078 EATR0156 EATR0795 GUP2498
GPAL/KE/6lIEATROl8 MHOM/KE/6l/EATR094 MHOM/KE/6l/EATR095 MHOM/KE/6l/EATRO% MHOM/KE/6l/EATRO97 MHOM/KE/6l/EATR0106 MHOM/KEMl/EATROll6 MHOM/KE/6l/EATROl56 MBOI/KE/ti/EATR0795 MHOM/KE/77iEATR02340
Tsetse MElll Man Man Man Man Mall Man Cattle Man
1%1 1%1 1%1 1961 1961 1%1 1961 1961 1964 1977
Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Yimbo, Alego, Samia,
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 GPAL/UG/6O/EATR03 MHOM/UGI8lIUTRO2 MHOM!UG/8l/UTR03 MHOM/UG/8l/UTR04 MBOYUG/88/IYOLWA96 MHOM/UG/BS/UGANDA MHOMIUGI88IUGANDA MBOYUG/88/IYOLWAl25 MBOIfLJG/SS/MELA27 MBOI/UG/88/IYOLWAl02 MHOM/UG/88/UGANDA MBOI/UG/88/MELA71 MBOYUG/88/PAPOL42 MSUS/UG/88/MELA PIG MBOYUG/88/MELA2 MBOI/UG/88/POYEMll MBOVUG/88/IYOLWAl53 MHOM/UG/88/UGANDA MHOM/UG/88/UGANDA MHOMIUGI88IUGANDA MHOM/UG/88/UGANDA
Man Tsetse Man Man Man Cattle Man Man Cattle Cattle Cattle Man Cattle Cattle m Cattle Cattle Cattle Man Man Man Man
1959 1960 1981 1981 1982 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988 1988
Busoga, Busoga, Busoga, Busoga, Busoga, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa, Iyolwa,
Zambian stocks 2194 2208 2210 2218 2222 2269 2274 2290
MHOMIZM/8UZl94 MHOM/ZM/82/Z208 MHOM/ZM/82/Z210 MHOM/ZM/82/Z218 MHOMlZMl82JZ222 MCAPlZMl83iZ269 MHOM/ZM/82/Z274 MHOM/ZM/8l/Z%
Man Man Man Man MkUl Goat Man Man
1982 1982 1982 1982 1982 1983 1982 1981
Chibale, Zambia Kasyasya, Zambia Kasyasya, Zambia Chibale, Zambia Kasyasya, Zambia Kasyasya, Zambia Kasyasya, Zambia Chibale, Zambia
A F
H
B C E G
from West Africa and were isolated both from humans and animals. As with group 3, these stocks represent West African “rhodesiense’‘-like trypanosomes. Group 7. This group contains a large
Kenya Kenya Kenya Kenya Kenya Kenya Kenya Kenya Kenya Kenya Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda Uganda
number of stocks isolated from animals (cattle and sheep), tsetse, and man from Kenya and Uganda. Group 8. The stocks contained within this group are all isolated from cattle in
OF T. b.rhodesiense
CHARACTERISATION
433
STOCKS
tem, based on similarities and differences in banding patterns generated by repetitive DNA probes, to characterise the relationships between trypanosome stocks (Hide et al. 1990). The main advantage of this system is that it is able to establish levels of identity and difference between trypanosome stocks, based on variation in multiple loci, without dependence on other biological information available on the stock. The sensitivity of this approach is enhanced by the use of a large number of characters FIG. 1. A typical Southern blot showing banding (bands) such that high degrees of similarity patterns produced when Hind111 digested trypanosome DNA was probed with pBE2. (A) A comparison in banding patterns reflect close genetic of Kenyan T.brucei stocks, TREU 869 (lane l), identity. It is not possible to place exact EATRO 156 (lane 3), GUP 1043 (lane 4), and stocks probabalistic values on the confidence with isolated from Zambia, Z 290 (lane 2) and Z 269 (lane 5). which genetic identity and difference can (B) A comparison of trypanosome stocks from Uganda. UG 14, 15, 16, and 17 (lanes 3-6, respec- be established, using this approach, as no tively) were isolated from man, and UG 12 and 13 information is available on rates of change (lanes 1 and 2, respectively) were human serum sen- of these banding patterns in trypanosome sitive trypanosomes isolated from animals. Sizes of field isolates. However, the sensitivity of some of the bands are given in kilobases for reference this approach was assessed by a comparipurposes. son, carried out blind, with isoenzyme and biological characteristics obtained from Uganda and are human serum sensitive other work (Maudlin et al., in preparation), (i.e., not human infective). using a collection of trypanosome stocks Group 9. The stocks contained within from Uganda (UGl-17). Complete agreethis group are isolated from pigs and cattle ment between these different approaches in Uganda and are human serum sensitive. was observed. This system is, therefore, capable of answering a number of imporDISCUSSION tant epidemiological questions which may In a previous study, we have used a sys- not be answerable using other approaches. B
A
123456
12345
1 2 3 4 5 6 7 6 ----------------------------------------,a ----------------I-----------------------~~ _____m-e--v-------------------,,,,,1,,-11------,,-,,,,,,,,,1,,,,,,,,,,~~ ___________--_-------------
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-
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910111213141516171819
202122232425262726293031323334353637363940 -
---------------s--w
-_-
-
-
---
-
-
-
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-
-
-
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-
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-
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--
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-
-
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-----------e-----35 -
--
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----80
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-
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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 -_-------_------_--_------------
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--------------------------------------m-7.2 --------------------------~~--~~--~--~~-65 --s----m
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--
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----
--
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--
----a,5 ---w-3,3
--
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--
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--
-----
5.2 6.7 5.5
i.:
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 collected 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 (classically 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 trypanosome stocks, using this repetitive DNA system, to investigate stocks isolated from the major sleeping sickness foci in Zambia, Kenya, and Uganda. 1
*
3
4 5
6
7 8
9
-
14
15 16171819
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 northward over the ensuing years (Ormerod 1961). It lies in a tsetse fly belt, predominantly 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 further 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 infection occurs within villages. In contrast a
20 2122
23 24 25 26 27 282930313233
34 3536
37 38 3940 I20 103
--
-----
10 11 12 13
of Trypanosoma brucei rhodesiense Stocks
Characterisation
---------
-
--
--
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_-
?eO 95
____
----____-----------_----
--
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---
,":
---------------------81 --------------__-
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-
-
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____
78 7.3 7.1
----
;:
--
-------------------------------------~~ -------------------------------------L2 ----------------------------------------4,5 ----------------------------------------4,3 ___-------------------------------
------,,
FIG. 4. As in Fig. 2 except that Hind111 digests of trypanosome DNA were hybridised to pBE2.
6
OF T.b.rhodesiense
CHARACTERISATION 1 2
3
4
5
6
7 8
9 10 11 121314
15 16 1718
19 20 2122232425
435
STOCKS
262728293031323334
35383738
39 40
---m----135 --------I23 t1.2
--
,--,,,,,104
--1--1----1--
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--
9.7 8.9
--
---w
-
--
___-
-
-----
---
-
---
-
s-e
---
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-
--
-
--_
-
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-----we--
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---------
----------------------a,*
--m-------w__ -----
-
-----
-------_
-
--
-----8.7
s--m
-----w--------s-----
--
-
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----
--------------mm--
----me, -----6.0 5.7 4.9
--------------------__------__-----
__e-----------_----_-__-----
---------_____ -m-m m-mm--m
-
--
“,‘: 3.5 -
-
-
-
2.8 2.4
FIG. 5. As in Fig. 2 except that XhoI digest2i of trypanosome DNA were hybl idised to Lambda 109.
different epidemiological profile of T. b. rhodesiense sleeping sickness was observed in the more northerly foci in Kenya and Uganda. In these areas the disease protile was very acute (MacKichan 1944) and a higher prevalence of disease was observed in Uganda, (Abaru 1985) and Kenya (Gibson 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 explaining these epidemiological differences (Ormerod 1961). In 1982, a sleeping sickness outbreak occurred in a small village, Kasyasya, in the North of the Luangwa Valley. This outbreak 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 responsible for the disease in Zambia? Secondly, what factors are responsible for the high incidence of disease in Kasyasya Village? To answer these types of questions, a number of techniques have been deployed to characterise trypanosome strains. Analysis of isoenzyme variation has been the main method of analysis of trypanosome stocks. Using isoenzyme analysis, Gibson et al. (1980) examined a number of trypanosome stocks isolated from the Luangwa Valley in 1971-1974. The isoenzyme patterns observed in the human infective trypanosomes were quite distinct from other stocks isolated in the Northern foci in Kenya and Uganda. More recently, Godfrey et al. (1990) have confirmed this general 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 relationships 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
.-.-. hlos El,
NIPA OURAM FOUNA ZENOU ELIANE 1871 2290
Group 1 Group 2 Grow 3
Gro”p
4
Group
5
:::: 2222 2269 2274 BUP2S40 GUP 2546 GUP 2560 IXIP 2560
r
NITR4OI12 TAEU la96 TREU 1096 TREU 1397 TREU 1395 TREU 1399
L
Group 6
AMA
q!!ij
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
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 isolated in the Kenya/Uganda area are as great as the differences between T.b.gambiense and T.b.rhodesiense. Thus, it seems unlikely that the Kenyan and Ugandan foci were established by a northerly spread of strains from Zambia as previously suggested (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 alongside stocks isolated from another area of Zambia (Chibale) . Therefore, there is nothing strikingly different about the stocks responsible for the Kasyasya epidemic and those found elsewhere in the Luangwa Valley. The reason for the high incidence in Kasyasya would appear to be due to a peculiarity in the epidemiological relationships of man, vector, and reservoirs rather than the appearance of a new strain. On the other hand, the stocks isolated from Kasyasya 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 transmitted around the village by close manfly-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 household as other isolates (for example GUP2590) and previously isoenzyme studies had shown 2208 to have an identical isozyme pattern to the other stocks from this household (Dukes et al. 1983), we conclude that this inconsistancy is due to the stock being wrongly coded in the time be-
T.b.rhodesiense STOCKS
437
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 Kasyasya. This has a pattern identical to three other stocks isolated from patients in the village, again suggesting the spread of a single trypanosome strain throughout this village. 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 sleeping 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, analysis of more recent stocks has shown that at least six trypanosome strains were circulating (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
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 (Maudlin 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 Welbum, in preparation). They had very similar 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 patterns thus confirming that domestic livestock act as a reservoir for sleeping sickness in this area. In addition to the Iyolwa T. b.rhodesiense stocks, group 7 also contains a large number 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 patterns 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
ET AL.
4, GUP 1301, and GUP 1052 have identical patterns and therefore the strain was present in the human population in Kenya in 1961and 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 contained the human-infective stock, STIB 386, which was isolated in West Africa but previously shown to have banding patterns indicative of East African origins (Hide 1988;Hide et al. 1990). Thus, the grouping of this stock remains stable in this analysis despite the addition 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 hypothesis that T.b.rhodesiense sleeping sickness arose in the Luangwa Valley and spread northward to the Kenyan/Ugandan foci (Ormerod 1961). This is further supported 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 relatively stable. We might have expected some degree of identity between the Zambian and Kenyan/Ugandan stocks had there been common origins. The origins of this divergence are unknown; it may be that trypanosome strains acquire human serum resistance (i.e., human infectivity) as an independent event arising from the background 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 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 Fairley for the manuscript. We especially thank Richard Cibulskis (University of Liverpool) for kindly providing computer programs, which have greatly assisted the cluster analysis, and Peter Dukes (University of Bristol) for helpful discussion.
REFERENCES AIJARU, D. E. 1985. Sleeping sickness in Busoga, Uganda, 1976-1983. Tropical Medicine and Parasitology 36, 72-76.
BARRY, J. D., CROWE, J. S., AND VICKERMAN, K. 1983. Instability of the Trypanosoma rhodesiense metacyclic variable antigen repertoire. Nature 306, 699-70 1. BUYST, H. 1974. The epidemiology, clinical features, treatment and history of sleeping sickness on the northern edge of the Luangwa Fly belt. Medical Journal
of Zambia 8, 2-12.
DUKES, P., SCOTT,C. M., RICKMAN, L. R., AND WuPARA, F. 1983. Sleeping sickness in the Luangwa Valley of Zambia. A preliminary report of the 1982 outbreak at Kasyasya Village. Bulletin de la Societe de Pathologie Exotique 76, 605-613. DUKES, P., RICKMAN, L. R., KILLICK-KENDRICK, R., KAKOMA, I., WURAPA, F. K., DE RAADT, P., AND MORROW,R. 1984. A field comparison of seven diagnostic techniques for human trypanosomiasis in the Luangwa Valley, Zambia. Tropenmedezin und Parasitology 35, 141-147. FOULKES,J. 1970. Human trypanosomiasis in Zambia. Medical
Journal
of Zambia 4, 167-177.
GIBSON, W. C., AND GASHUMBA, J. K. 1983. Isoenzyme characterisation of some Trypanozoon stocks from a recent trypanosomiasis epidemic in Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene 77, 114-l 18.
GIBSON,W. C., AND WELLDE, B. T. 1985. Characterisation of Trypanozoon stocks from the South Nyanza sleeping sickness focus in western Kenya. Transactions of the Royal Society of Tropical Medicine and Hygene 79, 671-676.
GIBSON, W. C., MARSHALL, T. F. DE C., AND GODFREY, D. G. 1980. Numerical analysis of enzyme polymorphism. A new approach to the epidemiology and taxonomy of trypanosomes of the subgenus Trypanozoon. Advances in Parasitology 18, 175245.
439
STOCKS
GODFREY, D. G., BAKER, R. D., RICKMAN, L. R., AND MEHLITZ, D. 1990. The distribution, relationships, and identification of enzymic variants within the subgenus. Trypanozoon Advances in Parasitology 29, l-74. HIDE, G. 1988. Variation in repetitive DNA in African trypanosomes. PhD. thesis, University of Edinburgh. HIDE, G., CATTAND, P., LE RAY D., BARRY, J. D., AND TAIT, A. 1990. The identification of Trypanosoma brucei subspecies using repetitive DNA sequences. Molecular and Biochemical Parasitology 39, 213-226. MACKICHAN, I. W. 1944.Rhodesian sleeping sickness in eastern Uganda. Transactions of the Royal Society of Tropical
Medicine
and Hygiene
38, 49.
OR~E~OD, W. E. 1961. The epidermic spread of Rhodesian sleeping sickness 1908-1960. Transactions of the Royal Society of Tropical Medicine
and Hygiene
55, 525-538. RICKMAN, L. R. 1974. Investigations into an outbreak of human trypanosomiasis in the lower Luangwa Valley, Eastern Province, Zambia. East African Medical Journal 51, 467-487. SAMBROOK,J., FRITSCH, E. F., AND MANIATIS, T. 1989. Molecular Cloning: A laboratory manual. Second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. STEPHENS,J. W. W., AND FANTHAM, H. B. 1910. On the peculiar morphology of a trypanosome from a case of sleeping sickness and the possibility of its being a new species (T.rhodesiense). Proceedings of the Royal Society, Series B 83, 28.
TAIT, A. 1980. Evidence for diploidy and mating in trypanosomes. Nature 287, 536-538. TAIT, A., BABIKER, E. A., AND LE RAY, D. 1984. Enzyme variation in T.brucei spp. I. Evidence for the subspeciation of T.b.gambiense. Parasitology 89, 311-326. TAIT, A., BARRY, J. D., WINK, R., SANDERSON,A., AND CROWE,J. S. 1985. Enzyme variation in T.brucei ssp. II. Evidence for T.b.rhodesiense being a set of variants of T.b.brucei. Parasitology 90, 89-100. TIBAYRENC, M., KJELLBERG,F., AND AYALA, F. J. 1990. A clonal theory of parasitic protozoa: The population structures of Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Trichomonas and Trypanosoma and their medical and taxonomical consequences. Proceedings of the National Academy of Sciences, USA 87, 2414-2418.
WURUPA, F., DUKES, P., NJELESANI, E. K., AND BOATIN, B. 1984. A “healthy carrier” of Trypanosoma rhodesiense: A case report. Transactions of the Royal So&sty of Tropical Medicine
and Hygiene
78, 349-350. Received 22 August 1990; accepted with revision 16 November 1990
