Molecular and Biochemical Parasitology, 39 (1990) 213-226 213 Elsevier

MOLBIO 01294

The identification of Trypanosoma brucei subspecies using repetitive D N A sequences

Geoff Hide 1, Pierre Cattand 2, Dominique LeRay 3, J. David Barry 4 and Andrew Tait 1 IWellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, Glasgow, U.K., 2World Health Organization,

Geneva, Switzerland; 31nstitute of Tropical Medicine 'Prince Leopold', Protozoology Laboratory, Nationalestraat, Antwerp, Belgium; and 4Wellcome Unit of Molecular Parasitology, Department of Genetics, University of Glasgow, Glasgow, U.K.

(Received 21 September 1989; accepted 19 October 1989)

We describe the use of repetitive DNA probes to characterise the relationships between different stocks of African trypano- somes representing the subspecies of Trypanosoma brucei. Probes derived from the ribosomal RNA genes (coding region and non- transcribed spacer) and another repetitive DNA sequence were used to characterise trypanosome stocks by Southern blotting. Numerical taxonomy methods applied to the resulting restriction enzyme patterns were used to derive a dendrogram depicting the relationships between the stocks examined. We show that three groups of West African human infective stocks can be distin- guished: firstly, a group containing exclusively T. b. gambiense; secondly, a group which is indistinguishable from animal isolates in West Africa; and thirdly, a single stock which is indistinguishable from East African T. b. rhodesiense. In addition, we observe that T. b. rhodesiense stocks from East Africa are indistinguishable from animal isolates from the same area. Finally, we show that a group of 7". b. rhodesiense stocks, isolated from a 1978 sleeping sickness outbreak in Zambia, are probably derived from a single parasite strain, and that this strain is distinct from T. b. rhodesiense parasites from Kenya and Uganda.

Key words: Trypanosoma brucei subspecies; Numerical taxonomy; Repetitive DNA; Ribosomal RNA genes; Speciation

Introduction

The classification and identification of African trypanosomes of the Trypanosoma brucei group [1] has been a major area of research for many years. T. b. brucei, T. b. rhodesiense and T. b. gambiense, the members of the T. brucei group of subspecies, are morphologically identical, but differ in features such as their host range, geo- graphical distribution and the pathological effects of the disease they cause. Four main criteria have been recognised which define the three subspe- cies. (1) Host range. T. b. brucei infects domestic and wild animals, while T. b. rhodesiense and T.

Correspondence address: Geoff Hide, Wellcome Unit of Molecular Parasitology, Dept. of Veterinary Parasitology, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, U.K.

Abbreviations: DE52, DEAE cellulose; SDM, semi-defined medium.

b. gambiense also infect man. (2) Disease in man. T. b. rhodesiense causes acute disease in man, while T. b. gambiense causes chronic disease. (3) Infectivity to laboratory rodents. Both T. b. rho- desiense and T. b. brucei are highly virulent, while T. b. gambiense is avirulent. (4) Geographical distribution. T. b. rhodesiense is found in East Africa, T. b. garnbiense in West and Central Af- rica and T. b. brucei throughout the tsetse region of Africa [1]. In most cases, exceptions to these criteria can be observed. For example, in the case of the host range criterion, it has been observed that human infective trypanosomes can also be found in animals [2-4]. Because of the difficulties in assigning definitive biological characteristics to each subspecies, much recent research has con- centrated on using biochemical and molecular approaches for the identification of the African trypanosomes. These approaches have, in a num- ber of cases, begun to elucidate further complex- ities, in the taxonomy and epidemiology of the

0166-6851/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

214

African trypanosomes, which may not have emerged from classical epidemiological studies. For example, isoenzyme and DNA polymorph- ism analysis carried out on West African human infective trypanosome stocks, traditionally class- ified as a single group (T. b. garnbiense) has es- tablished other animal hosts for these stocks [5-9] and shown that trypanosomes resembling T. b. rhodesiense can be observed within this group. [5,9 I.

We have set out to further investigate the tax- onomic and epidemiological relationships be- tween the T. brucei subspecies by examining DNA polymorphism within the ribosomal RNA genes and a trypanosome repetitive DNA sequence, pBE2 [10]. Using a numerical taxonomy ap- proach based on restriction enzyme banding pat- terns, we have grouped together trypanosome stocks with similar banding patterns, and we dis- cuss these groups in relation to the epidemiolog- ical characteristics of the trypanosome stocks used in the study.

Materials and Methods

Trypanosome stocks. Five collections of T. brucei spp. stocks were used for this study. These are summarised in Table I. In addition to these stocks, we also included one stock each of Trypanosoma vivax, Trypanosoma congolense, Trypanosoma equiperdum and Trypanosoma evansi.

Isolation of trypanosome DNA. Trypanosomes were grown either by passage through mice, us- ing standard procedures, or by culturing pro- cyclic forms in SDM-79 medium [11]. Trypano- somes were purified from blood by anion exchange on DE52 [12]. DNA was extracted from the trypanosomes using a standard procedure [13].

Isolation of phage lambda and plasmid DNA. Lambda DNA was prepared by a combination of a number of established methods [14]. Essen- tially, 1011 phage, which had been harvested by the plate lysate method, were used to inoculate a 100-ml liquid culture of host bacteria. Following lysis of this culture, the bacterial debris was spun out, the phage pelleted, and purified by caesium chloride step gradient centrifugation [14]. Fol-

lowing dialysis against 10 mM Tris, 1 mM EDTA pH 8.1, the phage were extracted in phenol, phenol/chloroform and chloroform to purify the DNA. The DNA was then precipitated with iso- propanol and resuspended in an appropriate buffer.

Plasmid DNA was extracted as previously de- scribed [14]. Purified DNA species were analysed by restriction enzyme digestion and agarose gel electrophoresis using standard procedures [14].

Construction and screening of genomic library. HindlII-digested trypanosome DNA was ligated into the phage vector arms of h NMl149, and packaged in vitro using packaging extracts pre- pared by the method of Grosveld et al. [15]. A library containing 108 recombinant phage was ob- tained.

Plasmid DNA, containing part or all of the try- panosome ribosomal repeat, was labelled with [32p]dCTP by nick translation [16]. DNA from recombinant phage was transferred to nitrocel- lulose by the plaque transfer method [17]. Hy- bridisation of the probe to the filters was carried out as previously described [14] at 65°C in the ab- sence of formamide. Three rounds of plaque pu- rification were carried out to ensure complete cloning.

Southern blotting. 3-5 txg of trypanosome DNA were digested with the appropriate restriction en- zymes and run on either a 0.4% or 0.6% agarose gel at 40 V overnight and transferred to nitrocel- lulose using standard procedures [18]. Filters were hybridised to the probe as follows: the baked fil- ters were rinsed in 4 × STE (1 x STE = 100 mM NaC1/10 mM Tris, pH 8.0/1 mM EDTA) and washed for 3 h at 65°C in 10 x Denhardt's solu- tion/5 x STE/0.1% sodium dodecyl sulphate (SDS)/0.1% sodium pyrophosphate. The filters were then prehybridised in the above solution containing 0.1 mg m1-1 sonicated boiled salmon sperm DNA at 65°C for 2 h. The filters were then hybridised overnight at 65°C in the above solu- tion containing 10% dextran sulphate with 0.1 mg m1-1 salmon sperm DNA containing 106 cpm of labelled probe (boiled). The filters were washed as follows: 4 × STE for 15 min at room temper- ature; 10 × Denhardt's solution/4 × STE/0.1%

TABLE I

Designation and origin of trypanosome stocks

215

Stock Other designation Host Date Location Cloned

Trypanosoma brucei brucei: East Africa

TREU869 GPAL/KE/69/EATRO 1583 Tsetse 1969 Kiboko, Kenya + TREU927 GPAL/KE/70/EATRO 1534 Tsetse 1970 Kiboko, Kenya + TREU981 GPAL/KE/69/K4 Tsetse 1969 Kiboko, Kenya + TREU975 GPAL/KE/69/K18 Tsetse 1969 Kiboko, Kenya + LUMP258 GPAL/KE/69/K9 Tsetse 1969 Kiboko, Kenya + EATRO427 MOVS/UG/60/EATRO 427 Sheep 1960 Uganda + EATRO227 MOVS/UG/60/EATRO 227 Sheep 1960 Uganda + EATRO1125 MTRG/UG/66/EATRO 1 1 2 5 Bushbuck 1966 Uganda +

Trypanosoma brucei brucei: West Africa

TREU1096 MBOI/NG/71/TREU 1096 Cattle 1971 Nigeria + TREU1097 MBOI/NG/71/TREU 1097 Cattle 1971 Nigeria + TREU1395 MBOI/NG/77/NITR 40/4 Ox 1977 Nigeria - TREU1396 MBOI/NG/77/NITR 40/17 Ox 1977 Nigeria + TREU1397 MSUS/NG/62/NITR 8/18 Pig 1962 Nigeria - TREU1399 MBOI/NG/66/NITR 30/97 Ox 1966 Nigeria + NITR40/12 GMOS/NG/70/NITR 40/12 Tsetse 1970 Nigeria +

Trypanosoma brucei rhodesiense: East Africa

EATRO605 MHOM/KE/61/EATRO 605 Man 1961 EATRO609 MHOM/KE/61/EATRO 609 Man 1961 EATRO120 MHOM/KE/61/EATRO 120 Man 1961 GUP788 MHOM/KE/61/EATRO 148 Man 1961 GUP795 GPAL/KE/61/EATRO 7 Tsetse 1961

Trypanosoma brucei rhodesiense: Zambia

GUP2540 MHOM/ZM/82/199 Man 1982 Zambia GUP2548 MHOM/ZM/82/221 Man 1982 Zambia GUP2560 MCAP/ZM/83/273 Goat 1983 Zambia GUP2590 MHOM/ZM/82/220 Man 1982 Zambia

Trypanosoma brucei gambiense

Nyanza, Kenya + Nyanza, Kenya + Nyanza, Kenya + Sakwa, Kenya + Nyanza, Kenya +

STIB386 MHOM/CI/78/TH114 Man 1978 Koudougou, Ivory Coast + ELIANE MHOM/CI/52/ITMAP 2188 Man 1952 Ivory Coast + MOS MHOM/CM/74/ITMAP 1787 Man 1974 Cameroun + MA MHOM/CG/74/ITMAP 1898 Man 1974 Congo + PA MHOM/CG/75/ITMAP 1843 Man 1975 Congo + BIM MHOM/CM/75/ITMAP 1789 Man 1975 Cameroun + SETI MHOM/CI/83/DAL 558 Man 1983 Daloa, Ivory Coast - NIPA MHOM/CI/83/DAL 654 Man 1983 Daloa, Ivory Coast - OUSOU MHOM/CI/82/DAL 494 Man 1982 Daloa, Ivory Coast - OURAM MHOM/CI/83/DAL 631 Man 1983 Daloa, Ivory Coast - LIGO MHOM/CI/84/DAL 655 Man 1984 Daloa, Ivory Coast - FOUNA MHOM/CI/83/DAL 602 Man 1983 Daloa, Ivory Coast - KOBIR MHOM/CI/82/DAL 503 Man 1982 Daloa, Ivory Coast - ZENOU MHOM/CI/83/DAL 625 Man 1983 Daloa, Ivory Coast - TOBO MHOM/CI/83/DAL 596 Man 1983 Daloa, Ivory Coast - MAGO MHOM/CI/83/DAL 645 Man 1983 Daloa, Ivory Coast - ISTI MHOM/CI/83/DAL 607 Man 1983 Daloa, Ivory Coast - A B B A MHOM/CI/83/DAL 626 Mann 1983 Daloa, Ivory Coast -

The stocks used in this study are listed and have been obtained from a number of different stabilate banks leading to a consid- erable heterogeneity in designation. The abbreviation used to designate the T. b. brucei and T. b. rhodesiense stocks are as fol- lows: EATRO, East African Trypanosomiasis Research Organisation; TREU, Trypanosomiasis Research Edinburgh University; LUMP, London University Medical Protozoology; NITR, Nigeria Trypanosomiasis Research; GUP, Glasgow University Proto- zoology. The T. b. gambiense stocks are designated individually by letters according to the system used at the Institute of Tropical Medicine (Antwerp) except for one stock. STIB 386, obtained from the Swiss Tropical Institute, Basel. The nomenclature rec- ommended by the World Health Organisation is also quoted for each of the stocks.

216

sodium pyrophosphate/0.1% SDS for i h at 65°C; 4 x STE/0.1% SDS for 30 min at 65°C, 2 x STE/0.1% SDS at 65°C for 30 min; 1 × STE/0.1% SDS at 65°C for 1 h and 0.5 × STE/0.1% SDS for 1 h at 65°C. The filter was then dried and auto- radiographed at -70°C.

Cluster analysis. The comparison of banding pat- terns between individual members of the collec- tion of stocks was carried out using cluster anal- ysis. The following precautions were taken when interpreting the banding patterns observed for each trypanosome stock. Each trypanosoma DNA sample was run more than once, and in some cases it was necessary to alter the gel conditions to resolve close bands. At least one and more usually two, 'standard' DNA samples were run on each gel to assist with the interpretation of the banding patterns. A number of exposures of the same autoradiographs were also made to allow correct detection of bands of different intensities. Partially digested DNA samples could be recog- nised by the appearance of a ladder of high-mo- lecular-size bands and by their irreproducibility. These samples were repeated until reproducible. Some of the probes used in the analysis produce bands of very high molecular size which could not easily be measured. In these cases, such bands were not included in the analysis. The band sizes shown in Figs. 4-7 represent the only bands used in the analysis. All bands between the extremes of size, indicted in these figures, which appeared on the autoradiographs were reproducible and included in the analysis. Examples of the DNA banding patterns obtained for a given exposure are shown in Fig. 3. The combined information from a number of such autoradiographs was used to assign the stock-specific banding patterns illus- trated in Figs. 4--7. The banding patterns ob- tained for each trypanosome stock were used to calculate the Jaccard coefficient [19], a measure of similarity, for all pairwise combinations of stocks. The Jaccard coefficient was calculated as the ratio of the number of bands present in both stocks to the sum of the number of bands exclu- sive to the first stock plus the number exclusive to the second stock plus the number of bands common to both stocks. The resulting matrices of pairwise similarity coefficients for each restriction

enzyme and probe were combined to produce a single matrix of the mean similarities between each pairwise combination of stocks. The similar- ity coefficients were then used to construct a den- drogram of relatedness of banding patterns using the Group Average Cluster analysis technique [20]. The results found using this cluster analysis algorithm were found to be comparable to that obtained from other such procedures for example the Complete Linkage analysis method (Hide, G., PhD. Thesis, University of Edinburgh).

Results

Cloning of the ribosomal RNA genes. A genomic library of trypanosome DNA was screened with plasmids containing parts of the ribosomal coding region, (pGH174 and pGH332; ref. 21) and a plasmid (pR4; ref. 22) containing the whole ri- bosomal repeat to obtain clones of the ribosomal coding region and the non-transcribed spacer. Eight recombinant phage were isolated from the library and characterised by restriction mapping. Three of these clones, X102, X105 and X109 had inserts of 10 kb and corresponded to the non- transcribed spacer region, while the five remain- ing clones, h50, X55, X59 h60 and X104, had 7.5- kb inserts and represented the coding region. The identity of these clones was confirmed by hybri- dising each clone back onto a Southern blot of pR4 (data not shown), h104 and X109 were used as standard probes for the coding region and non- transcribed spacer, respectively.

Structure of the ribosomal repeat. Southern blots of HindlII-digested genomic DNA from TREU869 were probed with pR4, X104 or X109. The probe pR4 identified four main restriction fragments of 10-kb, 9.5-kb, 7.5-kb and 2-kb (Fig. 1A); however, these did not correspond to the HindlII map proposed in previous work, [21], where two HindlII fragments of 10 and ll-kb were reported. Blots probed with X104 (coding region) identified two major fragments of 7.5 and 9.5-kb (Fig. 1B), whereas blots probed with h109 (non-transcribed spacer) showed a single major band of 10-kb (Fig. 1C). Thus it can be con- cluded that the 7.5-kb and 9.5-kb fragments rep- resent the coding region, and the 10-kb fragment

217

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1 0 9 . 5 7 . 5 9 . 5

7 . 5 1 0

ili!i!i!iii~iii!i~!ii!i!iii~! iii!ii!!!i!!ii~!ii!i~iiiii ............ ii~ii~il . . . . iiii!iii!i~i!!~iiiii~iiii

2 0 - iii!!i • ~iiiii iiill i

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Fig. 1. Southern blots of HindlII digests of total genomic DNA probed with (A) the complete ribosomal repeat, plasmid pR4, (B) the ribosomal structural probe, h104 and (C) the ribosomal spacer probe, h109. The sizes of the major fragments are indicated and are determined relative to standards run on the same gel. The trypanosoma stock or species from which the genomic DNA

was isolated are indicated above each track.

represents the non-transcribed spacer. Restric- tion mapping of pR4 showed that the size of the total repeat was 19.5-kb, confirming the previous size estimates of the ribosomal RNA gene repeat [22]. However , the sum of the fragments ob- served on the Southern blot of trypanosome DNA probed with pR4 was 29-kb. The simplest expla- nation for this discrepancy in size between the re- striction map of pR4 and the genomic DNA is that more than one type of repeat is present, as shown diagrammatically in Fig. 2. This model postulates

T y p e A t t t 9 . 5 1 0

T y p e B t I I i 7 . 5 2 1 0

1 0 4 t t

1 0 9 t I

Fig. 2. Diagrammatic representation of the two ribosomal repeats (Type A and Type B) deduced from Southern blots of HindIII digested genomic DNA. The vertical lines indicate HindlII sites and the figures refer to the size of the fragments (in kb) generated by HindlII digestion. The dark block indi- cates the ribosomal structural portion of the repeats. The HindlII fragments cloned into k NMl149 are indicated (104 and 109); these clones are referred to as transcribed region

(104) and non-transcribed region (109), respectively.

that the ribosomal genes consis{ of type A re- peats with two HindIII sites, yielding fragments of 10-kb (non-transcribed spacer) and 9.5-kb (the coding region plus a short section of the spacer), and type B repeats, which are similar to type A except for an additional HindlII site within the 9.5-kb fragment yielding the 7.5-kb and 2-kb fragments. The sizes of the inserts from the re- combinant phage obtained from the library screen and the data from the Southern blots, shown in Fig. 1A, are consistent with such a model. This model is confirmed by the hybridisation of h104 (the coding region probe) to HindlII fragments of 9.5 and 7.5-kb, while h109 (non-transcribed spacer) hybridized only to the 10-kb fragment (Fig. 1B and C). The 2-kb fragment which ap- pears on the blot shown in Fig. 1A would not be detected by either h104 or h109 if this model were correct; this is confirmed by the results shown in Fig. 1B and C.

Restriction enzyme polymorphism in the ribo- somal coding region. Variation in ribosomal re- peats was observed when Southern blots of D N A isolated from E L I A N E (T. b. gambiense) were probed with either h104 (Fig. 1B) or h109 (Fig. 1C). The coding region probe (X104) only shows the 9.5-kb band, while the spacer region probe (h109) shows a major fragment of 10-kb. This

218

TABLE II

Major coding region polymorphisms in type A and B repeats

Restriction fragments

9.5-kb (Type (A) + + + + + + 7.5-kb (Type B) + + + -

T. b. brucei East African 5 2 1 West African 6 0 1

Total 11 2 2

T. b. rhodesiense East African 5 0 0 Zambian 4 0 0

Total 9 0 0

T. b. gambiense

Total 6 5 6

Overall total 26 7 8

The restriction fragments obtained when HindIII genomic digests are hybridised with the structural ribosomal gene probe h104. Three types of patterns have been scored: (1) fragments of 9.5 and 7.5-kb showing comparable intensity of hybridisation (+ +/+ +); (2) fragments of 9.5-kb and 7.5-kb with the latter fragment showing a considerable lower intensity of hybridisation ( + + / + ) ; (3) a single fragment of 9.5-kb (+ + / - ) . The numbers shown refer to the number of stocks exhibiting different restriction fragment patterns.

suggests that ELIANE possesses only one type of ribosomal repeat, the type A repeat. This feature was investigated further as a potential marker for discriminating between T. b. gambiense and the other subspecies.

A total of 41 stocks of T. brucei were analysed by Southern blotting of DNA digested with HindlII, probed with h104, and scored for the presence of the 9.5-kb and 7.5-kb fragments. Three classes of banding pattern were observed: stocks which had both the 9.5- and 7.5-kb bands; stocks which had only the 9.5-kb band; and stocks which had both bands, but in which the 9.5-kb band was more intense than the 7.5-kb fragment. Table II summarises the data and shows that the frequency of each type of banding pattern varies between the groups of stocks analysed. The re- suits show that these banding patterns could not be used to discriminate between the T. b. gam- biense and T. b. brucei stocks, as all combina- tions of banding patterns could be found in both groups of stocks examined. A difference, how- ever, was seen in the frequency of each banding

pattern within the groups. In addition to the intense 9.5-kb and 7.5-kb

fragments identified on Southern blots by h104, a number of less intensely hybridising fragments were observed. The pattern of these fragments varied between different trypanosome stocks. 40 T. brucei stocks and a representative stock of each of T. evansi, T. equiperdum, T. vivax and T. con- golense were examined for DNA polymorphism with the restriction enzyme HindlII. The banding patterns for each stock are illustrated diagram- matically in Fig. 4.

Restriction enzyme polymorphism in the ribo- somal non-transcribed spacer region. Restriction enzyme digests (HindlII or XhoI) of DNA from the collection of trypanosome stocks were South- ern-blotted and probed with h109, the ribosomal non-transcribed spacer probe. Fig. 3 shows a tYP- ical autoradiograph of a Southern blot using this probe. The banding patterns of the HindIII-di- gested DNA are summarised in Fig. 5 and the patterns of the XhoI-digested DNA are shown in Fig. 6.

A

10

8

- m

_

1 2 3 4 5 6 7 8 9

B

1 2 3 4 5 6 7

12-

10-

5-

4-

219

_

Fig. 3. Autoradiographs of Southern blots showing typical banding patterns observed when (A) HindIII digests of DNA from various trypanosome stocks are probed with pBE2 and (B) XhoI digests of trypanosome DNA was probed with k109. The stocks illustrated are as follows: (A) lanes 1-9 respectively, OURAM, OUSOU, NIPA, NITR40/102, TREU1399, TREU1396, EATROl125, TREU927 and TREU975. (B) lanes 1-7, respectively, TREU1096, GUP795, EATRO609, EATRO605, TREU927,

EATROl125 and TREU981. The size markers given are in kb.

1 2 3 4 5 6 7 8 9 1011 12131415161710192091~223242526272029303132333435363738394041424344

10 i J

8.3

7,5

6.0

5,7

5.5

LO

4.5

4.5

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3.5

2.5

3.0

1,5

~ n N B U a n 1 H i ~ t o n i numnl ~ ~ l i u ~ u U ~ lu iJ ~ qlma ~ - - u ha l

Fig. 4. Diagrammatic representation of the restriction fragment patterns obtained by Southern blotting of HindlII digests of total genomic DNA from 40 stocks of T. brucei ssp and one stock each of T. vivax, T. congolense, T. equiperdum and T. evansi. The blots were probed with the ribosomal coding region probe (h104). The thickness of the bands indicated represents the strength of hybridisational signal. The stocks examined are as follows: (1) TREU869, (2) TREU927, (3) EATROl125, (4) TREU981, (5) LUMP258, (6) TREU975, (7) TREU1097, (8) TREU1399, (9) NITR40/12, (10) TREU1396, (11) TREU1096, (12) TREU1397, (13) TREU1395, (14) EATRO605, (15) EATRO609, (16) EATRO120, (17) GUP795, (18) GUP2540, (19) GUP2548, (20) GUP2560, (21) GUP2590, (22) STIB386, (23) ELIANE, (24) MOS, (25) MA, (26) PA, (27) SETI, (28) NIPA, (29) OUSOU, (30) OURAM, (31) LIGO, (32) FOUNA, (33) KOBIR, (34) ZENOU, (35) ISTI, (36) ABBA, (37) T. evansi, (38) T. equiper-

dum, (39) T. vivax, (40) T. congolense, (41) EATRO427, (42) EATRO227, (43) BIM, (44) GUP788.

220

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10.0

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8,4

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65

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m mm mm mm m Imm mm m mm mm mm mmmm m mm mm mm | m m mm mmmm m m mm m m m | = m m | | | m m m

M

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m ~

Fig. 5. Diagrammatic representation of the restriction fragment patterns obtained by Southern blotting of HindlII digests of total genomic DNA from 40 stocks of T. brucei ssp. and one stock each of T. equiperdum and T. evansi. The blots were probed with the non-transcribed ribosomal spaced probe, h109. The key to the stocks examined is listed in the legend to Fig. 4 for lanes 1-40. The stocks shown in lanes 41-44 of Fig. 4 were not examined with this probe. The numbers listed down the side of the figure refer

to the sizes of the fragments in kb.

T. congolense and T. vivax DNA did not hy- bridise with the non-transcribed spacer probe. This is clearly shown in Fig. 1C, where h104 was shown to hybridise to T. congolense DNA, but k109 showed no hybridisation. However, the other Trypanozoon species, T. evansi and T. equiperdum, did hybridize with this probe. The banding patterns observed (Figs. 4, 5 and 6), when DNA from these two species was digested with HindlII or XhoI, were similar to the band- ing patterns observed with the T. brucei stocks. Comparison of the minor banding patterns, in both HindlII and XhoI digests, shows that the differences between the T. evansi, T. equiperdum and T. brucei stocks are at a comparable level to the interstock differences within the T. brucei

Restriction enzyme polymorphism using pBE2. An uncharacterised trypanosome repetitive DNA probe, pBE2 [10], was used to probe Southern blots of HindlII digested DNA from the collec- tion of trypanosome stocks. A typical autoradi- ograph is shown in Fig. 3. The banding patterns observed with this probe are shown diagrammat- ically in Fig. 7. This probe did not hybridise with T. congolense or T. vivax DNA under the con- ditions of stringency used.

Cluster analysis of banding patterns. In order to analyse the banding patterns observed in the col- lection of trypanosome stocks using the three probes (109, 104 and pBE2), we used a cluster analysis technique to group together stocks of

species, similar patterns (see Materials and Methods). The

I 2 3 4 5 6 7 8 9 1011 1213141516171819 20 2122 2324 25 26 27 2~ 29 30 3132 33 34 35 3G 37 38 30 40

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11.2 10.,( im -- immm m im I m mt mmlm ~mlml INto m m m m mm imm miNi lm m Ira= IroN ~ m~ m ~ m m m m~ m l~t -- toNI

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Fig. 6. As legend to Fig. 5, but XhoI-digested trypanosome DNA was run instead of DNA digested with HindIfI.

12.0 10,3 "tO:O

9,9 9,S 9.4 8.7 8.1 7.8 7.3 7.1 6.7 6.2 ~ M 5.9 5,S 5,2

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l

m

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m m m ~ n ~ l m m m m m m I m l m

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Fig. 7. As legend to Fig. 5 but pBE2 used as a probe on HindIII digested trypanosome DNA.

221

results of this cluster analysis are presented as a dendrogram in Fig. 8. It can be seen from the dendrogram that the T. brucei stocks fall into four basic groups and the other Trypanosoma species each fall into separate groups. The stocks found in each of the four T. brucei groups are as fol- lows: Group 1. This group contains all of the Nigerian T. b. brucei stocks and four T. b. gambiense stocks. Group 2. This group comprises all of the East Af- rican T. b. brucei and all of the T. b. rhodesiense stocks with the exception of the T. b. rhodesiense stocks from Zambia. Group 3. The group contains, exclusively, T. b. gambiense stocks. Group 4. This group contains the Zambian T. b. rhodesiense stocks. The other species of the genus Trypanosoma were observed to be more dissimilar. T. evansi and T. equiperdum differ at the 56% level and T. con- golense and T. vivax differ at the 90-100% level of dissimilarity. The differentiation of these other species from T. brucei using these criteria must be taken with reservation, as only one stock has been examined for each species. The higher lev- els of differences between the T. brucei group of species and T. congolense or T. vivax is not sur- prising as these stocks are also morphologically distinct.

Discussion

Repetitive DNA probes have been used in a number of systems to investigate the relationship within and between groups of organisms. For ex- ample, the relationship between sibling species of Drosophila have been examined using the ribo- somal RNA genes, histone genes and non-coding repetitive DNA sequences [23,24], whilst 'molec- ular fingerprinting', using a highly repetitive DNA sequence [25,26], has been employed to identify individual or related humans for forensic analy- sis. The basic rationale for using repetitive DNA probes is that it is possible to examine the varia- tion at many loci simultaneously and this varia- tion can then be quantified to get an assessment of the relatedness of pairs of individuals to each other. In this work, we examined the restriction enzyme patterns found in members of a collec- tion of T. brucei stocks using probes derived from the ribosomal RNA genes (coding region and non- transcribed spacer region) and an uncharacter- ised trypanosome repetitive DNA sequence, pBE2.

The T. b. gambiense stocks, as defined by iso- lation from humans in West Africa, were found to fall into three groups in the cluster analysis. Group 1 contained the four trypanosome stocks, LIGO, KOBIR, OUSOU and ABBA. Group 2 contained a single stock, STIB386, from West Africa, alongside the T. b. brucei and T. b. rho- desiense stocks. All the other T. b. gambiense

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1097

A b b a - -

O u s o u L lgo - -

K o b i r - -

1396 - -

40112

1397 - -

1395 - -

1096

1 3 9 9

605 - -

609

869 - -

120

258

927

2 981

1125

975

795

386

E l i a n e

I s t i

S e t i N i p a

O u r a m

3 F o u n a - -

Z e n o u

M O S - -

PA

k 2 5 4 0

4 2548

2560

2590

T . e v a n s i

T . e q u i p .

T . c o n g .

T . v i v a x

25 50 75 100 I I I I

% D i s s i m i l a r i t y

Fig. 8. A dendrogram showing the groups obtained using cluster analysis to group together stocks with similar restric- tion banding patterns (see Materials and Methods). The groups obtained (1-4) are indicated and the criteria and methods used to identify these groups are discussed in the text. Details of the identity of the stocks can be found in Materials and Meth-

ods.

stocks were found to fall into Group 3. This lat- ter group consisted exclusively of T. b. gambiense stocks. The four Group 1 isolates, all collected in Daloa, Ivory Coast, are closely related to the seven West African T. b. brucei stocks isolated from cattle (TREU1096, TREU1097), Ox (TREU1395, TREU1396 and TREU1399), Pig (TREU1397) and tsetse (N40/12) in Nigeria. These Nigerian stocks have been previously de- scribed as T. b. brucei, on the basis of their sen- sitivity to human serum [8]. This suggests that the Group 1 T. b. gambiense stocks are host range

variants of the West African T. b. brucei stocks. Previous studies have also identified trypano- some stocks, isolated from man in West Africa, which did not conform to T. b. gambiense on the criteria of isoenzyme markers [5,28] or antigen gene probes [9,27]. Thus the evidence presented here and evidence from previous studies suggests that human infective variants of T. b. brucei exist in West Africa. These 'non-gambiense' human parasites bear the same relationship to West Af- rican T. b. brucei as T. b. rhodesiense does to East African T. b. brucei. Detailed analysis of case histories would be required to investigate whether acute sleeping sickness could be correlated with these 'non-gambiense' stocks. Older reports of acute sleeping sickness occurring in areas of gam- bian (chronic) sleeping sickness further support the view that 'T. b. rhodesiense-like' parasites could be found in West Africa [29,30]. The Group 2 7". b. gambiense stock, (STIB386), isolated from man in Daloa, falls into a large group of stocks which encompass all of the East African (Kenyan and Ugandan) T. b. brucei and T. b. rhodesiense stocks. This stock has been previously classified as a 'non-gambiense' stock by both isoenzyme [5] and antigen gene probe criteria [9]. The repeti- tive DNA data, presented here, shows that it is indistinguishable from East African T. b. rhode- siense or T. b. brucei stocks. In view of its human isolation it is likely, therefore, that this stock is an East African T. b. rhodesiense. Furthermore, this stock, as shown in trypanosome crosses, is able to exchange genes with another East African stock [10]. The Group 3 T. b. gambiense stocks consist of trypanosome stocks isolated between 1982-1984 in Daloa and also some 'classical' T. b. gambiense stocks isolated between 1952-1975 from the Ivory Coast, Cameroun and the Congo. In particular one stock, ELIANE, which was iso- lated from a woman living in Paris subsequent to living in the Ivory Coast, showed a classic chronic profile of sleeping sickness. This feature is one of the characteristics associated with sleeping sick- ness caused by T. b. gambiense; in addition, this group of stocks was found to have relatively ho- mogeneous DNA banding patterns, a feature consistent with other data that classical T. b. gambiense stocks are relatively homogeneous for other characters such as antigen gene comple-

223

ment [9,27] "and isoenzyme patterns [31]. Although being broadly in agreement with the

studies using the antigen gene probes [27] our re- suits differ in the details of a single stock exam- ined by both groups. The stock KOBIR, desig- nated as T. b. gambiense using the antigen gene probe criterion [27], was clearly found to be a 'non-gambiense' stock using our repetitive DNA criteria. The possibility of a mix-up of stocks in our laboratory can be ruled out, as we can indi- vidually identify any given trypanosome stock us- ing our repetitive DNA patterns. Thus, as no other stock we have examined has a pattern iden- tical to KOBIR, we must conclude that if a mix- up occurred, it must have been prior to arrival at our laboratory. The most likely explanation for this discrepancy is that the original isolate was a mixture of a gambiense and 'non-gambiense' try- panosomes, and that by chance during the pas- saging of this strain, two different lines, de- scribed by the same name, have been obtained.

We have suggested, along with others [9,27], that the group 1 West African Human stocks are West African equivalents of T. b. rhodesiense. The epidemiological importance of this becomes apparent when considered in the light of recent observations on an RNA transcript present in hu- man serum resistant T. b. rhodesiense stocks [32]. This transcript is absent from human serum-re- sistant T. b. gambiense stocks, leading to the sug- gestion that two mechanisms may be associated with human infectivity. If this is the case, then it may be important in the future to be able to dis- tinguish these two types of human infective try- panosomes which coexist in West Africa.

The East African (Kenyan) T. b. rhodesiense stocks all fall into Group 2. In addition to these stocks, this group contains the East African T. b. brucei stocks which are defined as non-human in- fective on the basis that they had been isolated from an area where there have been no reported cases of sleeping sickness. In many cases the hu- man infective stocks are more closely related to non-infective stocks than to other human infec- tive stocks. This supports the current view that T. b. rhodesiense is a host range variant of T. b. bru- cei [33]. The four Zambian T. b. rhodesiense stocks were isolated from an epidemic of sleeping sickness in the Luangwa Valley of Zambia in 1982

[34], and originated from different people in a single village, Kasyasya, in the north of the Luangwa Valley. The repetitive DNA results show that all four stocks are identical. This sug- gests that all of these particular infections were derived from a single parasite which may have spread through the village. The cluster analysis indicates that the trypanosomes from this region of Zambia are very different from those found in Kenya. Previous studies have shown that a num- ber of unusual isoenzyme patterns have been ob- served in trypanosomes from this area [31]. How- ever, more extensive analysis of stocks from this area will be required to clarify the relationships of the Zambian T. b. rhodesiense stocks com- pared with other T. b. rhodesiense stocks.

The dendrogram shown in Fig. 8 indicates that there is a clear difference between the stocks of West and East African T. b. brucei (and their hu- man infective homologues in groups 1 and 2). This observation conforms previous results using is- oenzyme variation [31] and restriction fragment length polymorphism in kDNA [35]. This dichot- omy may be due to sampling from extremes of a continuum, rather than indicating the presence of two distinct groups.

Thus, the work reported here, in conjunction with other molecular and biochemical studies, suggests that the classical classification of the T. brucei sub-species is no longer supported by all the information available. Furthermore, using the probes described here and previously [9,29,36], it is now possible to define trypanosome stocks into groups based on their degree of similarity and provide a revised classification of the T. brucei group. If such an analysis is valid, it would be possible using the polymerase chain reaction [37] to define trypanosome stocks without the need for large quantities of parasite material.

Acknowledgements

This work was supported by grants from the World Health Organization Special Programme (J.D.B., A.T., G.H.), the WeUcome Trust (A.T.) and NATO (D. LeR., A.T.). We would like to thank Cathy Harrison and Sue Haley for techni- cal assistance and Afshan Fairley for typing the manuscript. We are indebted to Dr. J. Wells for

224

supplying us with the pBE2 gene probe and to Dr. D.G. Godfrey for supplying us with the stocks of

trypanosomes from Zambia. J.D.B. is a Well- come Trust Senior Lecturer.

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