Isoenzyme comparison of Trypanozoon isolates from two sleeping sickness areas of south-eastern Uganda

Acta Tropica, 55(1993)97-115 © 1993 Elsevier Science Publishers B.V. All rights reserved 0001-706X/93/$06.00

97

ACTROP 00326

Isoenzyme comparison of Trypanozoon isolates from two sleeping sickness areas of south-eastern Uganda J . C . K . E n y a r u a, J . R . S t e v e n s b, M. O d i i t a, N . M . O k u n a a, a n d J.F. C a r a s c o c aUganda Trypanosomiasis Research Organization, P.O. Box 96, Tororo, Uganda, bl Rue du Puits des Esquilles, 34000 Montpellier, France, CDepartment of Biochemistry, Makerere University, P.O. Box 7062, Kampala, Uganda (Received 27 April 1993; accepted 14 June 1993)

The study characterized 151 Trypanozoon isolates from south-east Uganda by isoenzyme electrophoresis. Stocks were from a range of hosts, including man, cattle, pigs, dogs and Glossina fuscipesfuscipes; 104 isolates were from the Busoga area, 47 were from the Tororo district. Stocks were characterized on thin layer starch gel using eight enzyme systems: ALAT, ASAT, ICD, MDH, ME, NHD, NHI, PGM. Enzyme profiles were generally typical of East Africa; new patterns for ICD and ME were detected. Trypanosomes were classified on the basis of their profile by similarity coefficient analysis and the unweighted pair-group method using arithmetic averages (UPGMA). The majority of trypanosomes were classified in one or other of two genetically distinct groups which corresponded to the strain groups busoga and zambezi, both of which are associated with Rhodesian sleeping sickness in East Africa. Contingency table analyses indicated associations between certain isoenzymes of ICD and PGM, according to host and geographical origin. Significant relationships between trypanosome strain group and geographic origin were also demonstrated for some host groups. Key words: Trypanosoma brucei rhodesiense; Uganda; Isoenzyme; Zymodeme; Contingency table

Introduction

The current epidemic of rhodesiense sleeping sickness in south-east Uganda has persisted since 1976 (Abaru, 1985; Mbulamberi, 1989). The disease apparently spread northwards from its old endemic area near Busoga on the shores of Lake Victoria, reaching epidemic proportions around 1982 (Gibson and Gashumba, 1983; Mbulamberi, 1989). The causes of the present epidemic are unknown, but were probably the breakdown of tsetse control measures about 1972 and the subsequent spread of Glossina fuscipes fuscipes (Okoth and Kapaata, 1986). The problem was exacerbated by changes in agricultural practices, probably owing tO the political turbulence of the time, which encouraged the growth of bush in which flies thrived. In recent years, the disease has moved east until the Tororo district is now a major focus of the epidemic (Enyaru et al., 1992). Within this district, the many swamps Correspondence to." J.R. Stevens, UMR CNRS-ORSTOM 9926, Laboratoire G6n6tique Mol6culaire des Parasites et des Vecteurs, ORSTOM, 911 Avenue Agropolis, BP 5045, 34032, Montpellier, France. Fax: + 33 67547800.

98 and areas of riverine forest provide ideal tsetse habitat (Maudlin et al., 1990), and it is from this area that many of the stocks used in this study were isolated. The others were collected from the old endemic area of Busoga (Fig. 1). Previous isoenzyme characterization of trypanosomes from the region indicated that in the period 1976-1981 at least six zymodemes were circulating in man in Busoga (Gibson and Gashumba, 1983), including one previously sampled from patients from Busoga in 1959 (Gibson et al., 1980). More recently, Stevens et al. (1992) classified seven isolates from Busoga patients in four zymodemes, while studies by Maudlin et al. (1990) and Stevens et al. (1992) classified 44 stocks from Tororo district in twelve zymodemes. Thus, while several localized studies have been made, a broad comparative study of sleeping sickness in both the endemic Busoga area and the epidemic area of Tororo remained to be undertaken. The current study addressed this situation, and characterized, by isoenzyme electrophoresis, a large number of trypanosome isolates recently collected from both Busoga and Tororo. This allowed comparison of the trypanosome populations circulating in each area, in relation to previous work, and with reference to epidemiology, clinical disease and transmission cycles.

Materials and Methods

Sample origins Stocks were collected from patients, domestic animals (cattle, pigs and dogs) and tsetse flies (G.f.fuscipes) in villages in the Busoga, Tororo and Mukono areas (Fig. 1)

Rhodesian ttypanosorniasisarea

Fig. 1. Map showingthe distributionof Rhodesiansleepingsicknessin south-eastUganda.

99 of south-eastern Uganda between 1988-1992, as part of a long running sleeping sickness surveillance project by the Uganda Trypanosomiasis Research Organization (UTRO). Other isolates were collected from patients attending the sleeping sickness hospitals at Busoga and Tororo. The origins of the 151 Trypanozoon isolates are given in Table 1; full details of these stocks are available from J.C.K. Enyaru at UTRO.

Trypanosomes Trypanosomes were isolated from patients and infected domestic animals by the direct inoculation of blood and/or cerebrospinal fluid intraperitoneally into mice. The macerated salivary glands of infected tsetse were similarly directly inoculated. Stabilates were prepared from the infected blood of these laboratory mice with 7.5% glycerol and 5 lag ml-1 heparin, prior to cryopreservation in liquid nitrogen. To obtain sufficient parasites for isoenzyme electrophoresis, stabilates were thawed and passaged once, or twice more through laboratory mice, prior to a final passage in laboratory rats. Some stocks were cloned at the first passage after thawing. Parasitaemias were determined by examination of tail blood using the 'matching' method (Herbert and Lumsden, 1976). Rats were exsanguinated by cardiac puncture at a parasitaemia of = > 107.5 trypanosomes ml- ~ Trypanosomes were separated from rat blood using a DEAE-cellulose column (Lanham and Godfrey, 1970). After centrifugation, the soluble extracts for enzyme electrophoresis were prepared using the method of Gibson et al. (1978).

Isoenzyme electrophoresis Isolates were characterized by electrophoresis on thin-layer starch gel (TSGE). The following seven enzymes were examined as described previously (Gibson et al., 1978, 1980): alanine aminotransferase (ALAT, EC 2.6.1.2), aspartate aminotransferase (ASAT, EC 2.6.1.1), isocitrate dehydrogenase (ICD, EC 1.1.1.42), malate dehydrogenase (MDH, EC 1.1.1.37), "malic" enzyme (ME, EC 1.1.1.40), phosphoglucomutase (PGM, EC 2.7.5.1), nucleoside hydrolase (substrate: inosine, NHI, EC 3.2.2.1). A second nucleoside hydrolase (substrate: deoxyinosine, NHD, EC 3.2.2.1) was examined using a modified version of the method of Stevens et al. (1992). NHI has been previously labelled as NH (Gibson et al., 1978, 1980; Lanham et al., 1981; Stevens et al., 1989; Godfrey et al., 1990). Some modifications to published methods were necessary: PGM gel buffer was prepared using 3 ml of tank buffer diluted to 50 ml with distilled water. Gel buffers were similarly prepared from a volume of tank buffer diluted to 50 ml for the following: ICD (8 ml), ME (5 ml), M D H (5 ml), NHI (3 ml), N H D (3 ml). Gels were run at 250 V for 3 h.

Contingency table analysis Contingency table analyses were performed to investigate the distribution of isolates in relation to strain group, and certain patterns of ICD and PGM, host, and geographical origin. Analyses were performed by X2, calculated according to standard statistical methods. However, where cell totals were five or less, analyses were

100 TABLE 1 Isolate origins Zymodeme

Year~

Host type

O r i g i n o f isolate Busoga

17 181b 313 319 322 347 357b 403 404 405 406 408

la 181a 239 309 351 356 357a 402 409

314a

Man Man Man Man Man Man Man Man Man Man Man Man

8 2 0 2 2 0 0 1 1 0 0 0

5 0

1988 1991 1990,91 1991 1990 1991 1990 1990 1990,91 1989 1990,91 1991 1990-92 1991,92 1989 1991 1991 1991,92

1 1 20 0 1 1 1 1 0 1 3 1 3 3 1 1 1 2

0 0 1 l 0 0 0 0 2 0 2 0 1 1 0 0 0 1

-

G.f.fuscipes

1991 1990 1989-91 1991 1991 1991

0 1 3 1 0 1

1 0 13 0 1 0

0 0 0

Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic

1991 1991 1990 1991 1989,91 1989 1992 1991 1989,91 1991 1990

1 2 1 1 1 1 1 7 2 1 2

0 0 0 0 5 0 0 1 0 0 0

-

Man Domestic Man Domestic Man Domestic Man Domestic Man Domestic Man Domestic Man Domestic Man Domestic Man Domestic

animal animal animal animal animal animal animal animal animal

Man Man

G.f.fuscipes 407

lb lc 18 237 304 314b 346 352a 412 413 414

Mukono

1988-91 1990 1990 1988 1990 1990 1991 1991 1990 1990 1991 1991

Gf.fuscipes 401

Tororo

Man

animal animal animal animal animal animal animal animal animal animal animal

1 0 0

1 l 0 0

1 1 1

-

-

101 TABLE 1 (continued) Zymodeme

Host type

Yeaff

Origin of isolate Busoga

Tororo

Mukono

415 416 417 418 419 420 421 426

Domestic animal Domestic animal Domestic animal Domestic animal Domestic animal Domestic animal Domestic animal Domestic animal

1990 1991 1989,90 1991 1990 1992 1991 1991,92

1 3 2 1 1 0 0 1

0 0 0 0 0 1 1 3

-

410

Domestic animal

G.f.fuscipes

1991 1991 1991 1991

I 1 2 0

0 0 0 0

0 1

G.f.fuscipes G.f.fuscipes G.f.fuscipes G.f.fuscipes G.f.fuscipes G.f.fuscipes G.f.fuscipes

1990 1991 1990 1990 1990 1991 1990

I 0 1 I 1 0 0

0 0 0 0 0 0 1

0 1 0 0 0 1 0

G.f.fuscipes 411

125 330 352b 422 423 424 425

Domestic animal

aYear, 1988-91 signifies the four years between 1988 and 1991 inclusive; 1989,91 signifiesjust the two years shown.

performed using a r a n d o m i z a t i o n p r o g r a m ( A L L O C 1 . F O R , J.R.S.). F o r each analysis 10 000 reassortments were made.

Numerical analysis To evaluate t a x o n o m i c relationships within the dataset, a phenetic analysis was performed. A d e n d r o g r a m was constructed by the ' U n w e i g h t e d P a i r - G r o u p M e t h o d using A r i t h m e t i c Averages' (Sokal a n d Michener, 1958) from a distance m a t r i x calculated using a similarity coefficient m e t h o d (Jaccard, 1908; Stevens a n d Cibulskis, 1990). Similarity values were calculated using all isoenzyme b a n d s for each enzyme between each pair o f zymodemes; a t a x o n o m i c c o m p u t e r package (TAXO, E. Serres a n d F. Kjellberg, C N R S Montpellier) was used to p r o d u c e the d e n d r o g r a m .

Results

Enzymes E n z y m e p a t t e r n s a n d strain groups were for the most part as described by G o d f r e y et al. (1990) a n d Stevens et al. (1992). However, two new patterns were discovered; these were I C D - 5 (Fig. 2) a n d M E - 2 9 (Fig. 3).

102

I

3

2

S

Fig. 2. The banding patterns obtained with ICD. Pattern numbers according to Godfrey eta]. (1990).

New pattern: ICD-5.

Ill

1

2

10

22

29

Fig. 3. The banding patterns obtained with ME. Pattern numbers according to Godfrey et al. (1990). New pattern: ME-29.

Classification of isolates Profile numbers were allocated (Table 2), as far as possible, according to Godfrey et al. (1990) and Stevens et al. (1992). When a match to previously identified zymodemes could not be made, a new profile number was allocated. To aid comparison of these new zymodemes with those previously published, a 'best match' is also given (Table 2) based on the criteria of Gibson et al. (1980), Godfrey et al. (1990) and Stevens et al. (1992). The enzyme ME was not used by Stevens et al. (1992); thus for profiles which are identical to a previously published zymodeme apart from the ME pattern, the profiles in the current study are differentiated by an a, b or c suffix, e.g. Zla, according to the ME pattern. The 151 Trypanozoon isolates were classified into 52 zymodemes (Tables 1 and 2). Five out of eight of the enzymes examined showed variations in their electrophoretic patterns (Table 2). The isoenzyme results show an immense diversity of zymodemes circulating in man, domestic animals and tsetse flies (Table 1). The 82 isolates from humans were separated into 24 zymodemes, of which 15 had been described before; nine had previously unrecorded combinations of isoenzyme patterns and were thus allocated new profile numbers. Zymodemes belonged to either the busoga* or zambezi strain group (Gibson et al., 1980), both of which are * Strain group names are given in italics to avoid confusion, primarily between the busoga strain group and the Busoga area; we do not presume to indicate that such strain groups are recognised Linnean taxonomic classes.

103 TABLE 2 Isoenzyme patterns Zymodeme

BM a

ALAT

ASAT

PGM

ICD

MDH

NHI

NHD

ME

la lb lc 17 18 125 181a 181b 237 239 304 309 313 314a 314b 319 322 330 346 347 351 352a 352b 356 357a 357b 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426

4 I 1 31 22 125 210 210 237 210 1 1 4 1 66 22 38 149 210 210 210 210 31 -

2 2 2 2 2 2 10 10 10 I0 10 2 2 2 2 2 2 2 10 10 10 10 10 10 10 10 10 2 10 10 l0 l0 10 10 10 10 10 l0 I0 l0 10 10 2 l0 10 2 I0 l0 4 2 10 10

I 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 I 1 1 1 1 I l 1 1 I 1 l 1 1 l 1 l 1 1 l l l 1 1 l 1 1 1 l

1 1 1 3 1 2 1 1 3 1 1 1 1 1 1 3 1 3 2 1 I 1 1 1 1 1 3 3 4 3 3 4 4 4 3 4 2 1 3 l 1 2 2 2 3 4 4 3 1 2 I 4

1 1 1 3 3 1 1 1 2 I 3 1 1 1 I 1 3 2 2 1 1 1 1 3 3 3 3 3 3 3 ! I 1 1 3 3 2 2 l 2 2 1 2 2 2 3 1 1 l 2 5 3

1 1 1 1 1 1 1 I 1 1 l I 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 ! 1 1 l 1 I I l 1 1 1 l 1 1 l 1 l 1 1 l 1 1 1 l

1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 I 1 1 I 1 1 1 1 1 1 l l 1 l 1 1 1 1 1 1 1 1 1 1 1 1 I I l 1 1 1 I 1

1 l 1 1 4 1 3 3 1 1 4 3 4 5 5 3 3 1 1 2 4 5 5 1 3 3 1 5 4 5 1 5 1 4 3 1 5 5 4 l 4 l 3 4 5 3 3 5 5 5 4 4

1 25 10 I 1 10 1 10 25 1 1 10 25 1 25 I I 1 25 I 1 1 25 I l 10 1 1 22 l 10 2 l l l 1 1 I 1 l 1 2 10 I 1 1 1 25 10 1 25 1

216

149 216 39 149 237 34 216 39 -

a B M , best m a t c h a c c o r d i n g to G o d f r e y et al. (1990) a n d S t e v e n s et al. (1992).

104

associated with rhodesiense trypanosomiasis; Z405 was intermediate between zarnbezi and kiboko (Gibson et al., 1980). For isolates with previously undescribed profiles, a 'best match' with the results from previous studies was made (Table 2, see above). The classification of all zymodemes was further reinforced by numerical analysis (see below). The 57 Trypanozoon isolates from domestic animals (cattle, pigs and dogs) were placed in 30 zymodemes, thus showing a higher degree of genetic diversity than that found in human isolates. Fifteen of these zymodemes had been previously described, while the remaining 15 were allocated new profile numbers. Trypanosomes in nine of the 30 zymodemes, which included stocks from nine cattle, four pigs and a dog, were also found in man (Table 3). Isolates in two zymodemes found in tsetse flies also occurred in cattle in Busoga. Of the 15 new zymodemes detected in domestic animals, five included, or were

TABLE 3 Zymodemes found in both man and animals Zymodeme

Loc.

Host man

cattle

la

Busoga

1

181a

Busoga Tororo

20 1

239

Busoga

1

1

309

Busoga

1

1

314a

Busoga Tororo

1

Busoga Tororo

2

356

Busoga Tororo

3 2

357a

Busoga Tororo

3 1

401

Busoga Tororo

3 13

402

Busoga

1

407

Busoga Tororo

1 1

409

Busoga Tororo

1

351

pig

dog

1

1

1

1

G.f.fuscipes

105 represented uniquely by, cloned stocks; five of the stocks in previously identified zymodemes were also cloned. Twelve Trypanozoon isolates from G.f.fuscipes were allocated to twelve different zymodemes, indicating a particularly high level of genetic diversity in trypanosomes isolated from tsetse flies. Only four zymodemes had been previously identified, while eight new combinations of isoenzyme patterns were discovered; these included one profile from a cloned stock. Three of the zymodemes from tsetse trapped in the Busoga area were also found in man at both Busoga and Tororo.

Distribution of enzyme patterns Certain trends in the distribution of zymodemes were noted in relation to host and site. These trends appeared largely related to pattern differences in the enzymes PGM and ICD, and, given their importance in previous studies of T. brucei spp, their distributions are investigated in some depth. Of the 82 isolates from humans, 50 were from the Busoga area, while 32 were from Tororo. In these stocks the combination of homozygous patterns PGM-1 and ICD-1 was found in 50% of isolates from Busoga and 19% from Tororo (Tables 1 and 2). Human isolates with the apparently heterozygous combination of PGM-3 and ICD-3 accounted for 28% of isolates from Busoga and 56% from Tororo. The predominantly West African combination of PGM-2 and ICD-2 (Godfrey et al., 1990) occurred in trypanosomes from a range of hosts, including five cattle and a dog from Busoga, and in an isolate from a tsetse fly from nearby Mukono district (Tables 1 and 2); however, it was not detected in man. In isolates from domestic animals, the combination of patterns PGM-1 and ICD1 was 33% in Busoga and 14% in Tororo (Tables 1 and 2), showing a similar trend with those isolates from man. The combination of PGM-2 and ICD-1, not previously described from Busoga, was isolated from three cattle and a tsetse fly in that area.

Contingency table analyses Contingency table analyses were performed to investigate the distribution of isolates according to host and geographical origin, in relation to (a) strain group, (b) certain patterns of ICD, and (c) patterns of PGM. Several analyses were performed, of which those producing significant X2 results, and those producing non-significant ~2 results of some potential epidemiological value are presented here. Given the unevenness of sampling from the two sites, such an approach was necessary if comparisons were to be valid. Stocks isolated from patients were compared according to strain group and geographical origin (Table 4). Stocks in zymodemes classified as zambezi were isolated significantly (p<0.05) more often from the Busoga area than from the Tororo district; stocks classified as busoga were approximately evenly distribution between the two locations. For isolates from domestic animals, the distribution of patterns ICD-1 and ICD2 at Busoga and Tororo did not show any significant association (Table 5). A similar comparison was not possible for isolates from man as ICD-2 was not detected. Stocks possessing patterns ICD-1 and ICD-3 were sufficiently numerous to allow three categories of analysis in relation to geographical origin; these were isolates

106 TABLE 4 Human isolates Location

Strain group

Total

busoga

zambezi

Busoga Tororo

23 22

27 9

Total

45

36

50 31

X2=4.83; Sig. (p<0.05). TABLE 5 Domestic animal isolates Location

ICD pattern

Total

ICD- 1

ICD-2

Busoga Tororo

19 3

13 0

Total

22

13

32 3

Z2= 1.94; Non-Sig.

from patients (Table 6a), domestic animals (Table 6b) and a pooled group of stocks from man, domestic animals and tsetse flies (Table 6c). Human isolates showed a significant association (p<0.05) between ICD pattern and place of origin (i.e., Busoga or Tororo), which gave an identical match to the distribution of human stocks in relation to strain group (Table 4). Isolates from domestic animals also showed significant association (p<0.025) between ICD pattern and geographical origin (Table 6b). However, the basis of the association appeared different, and was due to an abundance of the homozygous pattern ICD-1 in Busoga, and a lack in Tororo; after accounting for sample bias (Busoga 30:14 Tororo), ICD-3 was seen to be over-represented at Tororo. The pooled group (Table 6c) showed no significant association between ICD-1 and ICD-3, and isolate origin. Stocks possessing patterns PGM-1 and the heterozygous PGM-3 were also sufficiently common to allow three categories of analysis in relation to geographical origin; these were isolates from patients (Table 7a), domestic animals (Table 7b) and a pooled group of stocks from man, domestic animals and tsetse flies (Table 7c). TABLE 6 Distribution of ICD patterns I and 3 at Busoga and Tororo

(a) (b) (c)

Host group

n

Z2

Significance

Human Domestic animals Pooled isolates

81 44 133

4.83 6.70 0.01

p < 0.05 p < 0.025 Non-sig.

107 TABLE 7 Distribution of P G M patterns 1 and 3 at Busoga and Tororo

(a) (b) (c)

Host group

n

Z2

Significance

Human Domestic animals Pooled isolates

77 41 124

7.23 0.29 5.68

p < 0.01 Non-sig. p < 0.025

The analysis of PGM patterns was extended further by combining the number of stocks possessing either of the two heterozygous patterns PGM-3 and PGM-4, which both shared a common band with PGM-1. These were then compared (Table 8a, b, c) with the distribution of the homozygous pattern PGM-1 for three categories of analysis as previously described. The second homozygous pattern, PGM-2, was not found frequently enough in any host group to permit a meaningful association analysis. Human stocks showed significant associations between PGM pattern (PGMI/PGM-3, p < 0.01; PGM- 1/PGM-3 + PGM-4, p < 0.005) and isolate origin (Tables 7a, 8a). Isolates from domestic animals showed no significant association between PGM pattern and geographical origin (Tables 7b, 8b). The pooled groups of stocks from humans, domestic animals and tsetse flies (Tables 7c, 8c) showed significant associations (PGM-1/PGM-3, p<0.025; PGM-1/PGM-3+PGM-4, p<0.005) between PGM pattern and the geographical origin (i.e., Busoga or Tororo) of isolates.

Numerical taxonomy The groupings formed in the dendrogram (Fig. 4) corresponded closely to the strain groups defined by Gibson et al. (1980) and Godfrey et al. (1990); these were busoga, zambezi, kiboko and bouaflL The relative positioning of these groups in the dendrogram was also in good agreement with the positioning of corresponding groups in a recent broad taxonomic study of Trypanozoon by Stevens and Godfrey (1992). Notably, busoga and zambezi, which contain most zymodemes associated with rhodesiense sleeping sickness in East Africa, were discrete and well separated, being only 55% similar. All, but one, zymodemes from man were classified in these two groups. The busoga strain group showed some affinity with a small group of four possible bouaflb zymodemes, a group generally associated with animal trypanosomiasis in west and central Africa, but known to occur in Uganda, and to be capable of infecting man (Maudlin et al., 1990; Stevens et al., 1992). Other bouaflb zymodemes were less similar to busoga, TABLE 8 Distribution of P G M patterns 1 and 3 + 4 at Busoga and Tororo

(a) (b) (c)

Host group

n

g2

Significance

Human Domestic animals Pooled isolates

81 48 137

8.75 1.79 9.10

p < 0.005 Non-sig. p < 0.005

108 SIMILARZTY - JACCJ~D*S DZSTANCE X.O

QiS

0.S

0.?

01~

O;S

0|~,

356 401. 410 403 426 304 357a 357b 409 322

~.]

1B 420 Bu

130 17

],

' ~

402 404

I

'

419 411

i

o 418 424 414 412 43.5

181a 351

3~2a ~47 413 407 408 4Z1

442026- - 1 ~52b

3°,

1

ZBlb 31g la lc

-

-

lb 313 314b

314a 423 405 416

346

I

,3,

i

425 417

~

I .....

Fig. 4. Dendrogram constructed by UPGMA from a similarity coefficient matrix. Similarities between zymodemes were calculated by averaging the Jaccard coefficients from seven enzyme systems. Bu, busoga; Bo, bouafld; Z, zambezi; K, kiboko; X, mixed bouaflO/busoga group.

109 and were placed well apart with a single busoga zymodeme which possessed a previously unrecorded ICD pattern. Also placed apart were three zymodemes of kiboko, an East African strain group generally associated with animal trypanosomiasis in Kenya (Gibson et al., 1980, 1985; Godfrey et al., 1990; Stevens and Godfrey, 1992); these contained stocks from domestic animals, tsetse and man.

Analysis of numerical taxonomy groupings From the groupings defined by the dendrogram it is apparent that at Busoga, zymodemes in the busoga and zambezi strain groups were sampled approximately equally from man (Table 9a), while at Tororo more, though not significantly (~2), zambezi than busoga zymodemes were sampled (Table 9a). This result appears to conflict with the strain group/origin analysis of human stocks (Table 4) in which the number of zambezi isolates sampled from the Tororo area is much lower than the number of busoga isolates collected at Tororo; moreover, the contingency table (Table 4) indicates, significantly, a different relationship between the geographical origin of a human isolate and its isoenzyme strain group from that suggested by Table 9a. The differences between these two conflicting results can be explained when the numbers of stocks within zymodemes are taken into account, as the distribution of stocks among zymodemes is biased, a few zymodemes (Z17, Z181a, Z401) being represented by a large number of isolates. However, a review of the collection dates (Table 1; exact dates available from J.C.K. Enyaru) of isolates shows that these results are not the product of biased sampling, i.e., at just one time point from a single group of infected patients; rather stocks were collected over a period of 18 months for Z181a, up to 36 months for Z17. Over this time isolates included in this study from these three zymodemes were collected on the same day and medical

TABLE 9 Geographic distribution of zymodemes

(a) Human Location

Busoga Tororo Mixed

Strain group

busoga

zambezi

5 ! 4

5 7 1

(b) Pooled Location

Busoga Tororo Mixed

Strain group

busoga

zambezi

9 3 7

12 5 5

110 centre on only four occasions; moreover, patients travelled to medical centres from different locations. When data from all hosts were pooled, zymodemes from both Busoga and Tororo were distributed approximately evenly among the busoga and zambezi strain groups (Table 9b). Trypanosomes of seven busoga zymodemes were sampled from both Busoga and Tororo, while nine were found only at Busoga and three only at Tororo; five zambezi zymodemes were sampled from both areas, while twelve were sampled only from Busoga, with five occurring only at Tororo (Table 9b). This even distribution is in agreement with the contingency table analysis of pooled data for ICD patterns 1 and 3 (Table 6c), which appear to serve as the best strain group markers (Tables 4, 6a).

Discussion

The possible existence of two clinically different forms of rhodesiense sleeping sickness, acute and semi-acute, occurring in different regions within East Africa has been well documented (Ormerod, 1961, 1963; Apted, 1970). Moreover, there is evidence that the southern, Zambian form is the least acute and is characterized by few and mild symptoms (Ormerod, 1961, 1963; Apted, 1970; Buyst, 1974) with the presence of apparently healthy human carriers (Wurapa et al., 1984). The geographical locations of these two clinical types can be matched to the locations of two genetically distinct strains of parasite (busoga and zambezi) as shown by several studies using isoenzymes (Gibson et al., 1980; Godfrey et al., 1990; Mihok et al., 1990; Stevens and Godfrey, 1992) and restriction fragment length polymorphisms (RFLP) (Borst et al., 1981; Gibson et al., 1985; Hide et al., 1990, 1991). The strain group busoga appears to be associated with the epidemic northerly areas, while the zambezi group is associated with the southern semi-acute form of the disease. More recently, clinical and epidemiological differences have been noted for some rhodesiense sleeping sickness patients in the Busoga area, and a semi-acute form of the disease has been detected in contrast to the classically acute type (Smith, D.H., personal communication). Various hypotheses regarding the origins and pattern of spread of the two types have been proposed (Ormerod, 1961; Gibson et al., 1980; Godfrey et al., 1990), and despite the somewhat conflicting conclusions of these hypotheses, Uganda consistently appears as the main location where the two clinical forms of East African sleeping sickness overlap. Indeed, historically, it was also the place where the distributions of T.b.rhodesiense and T.b.gambiense overlapped (Robertson and Baker, 1958; Apted, 1970), and recent reports (Bailey and Smith, 1992; Enyaru et al., 1993) indicate a resurgence of T.b.gambiense sleeping sickness in the northwest of the country. The current study confirms the existence of at least two genetically distinct types of T.b.rhodesiense infecting man in south-east Uganda as previously proposed by Gibson et al. (1980), Godfrey et al. (1990), Hide et al. (1990, 1991) and Stevens and Godfrey (1992). The difference may be accounted for by different genetic forms of the infecting trypanosomes; indeed, for the most part, trypanosomes from the two sleeping sickness loci of Busoga and Tororo are genetically different, and the two strain groups can be readily distinguished by the use of isoenzyme markers.

Ili

The majority of isolates in the current study had typical East African enzyme patterns (Gibson et al., 1980; Gibson and Gashumba, 1983; Tait et al., 1985; Godfrey et al., 1990; Stevens et al., 1992). The PGM-1/ICD-I combination, characteristic of the zambezi strain group (Gibson et al., 1980), was predominant in stocks from the Busoga area; the PGM-3/ICD-3 combination, characteristic of the busoga strain group (Gibson et al., 1980), which was previously dominant in the Busoga area (Gibson and Gashumba, 1983) was now predominant at Tororo. The change in frequency and the apparent movement of genetic types at the two sites, suggests that the new focus of infection in Tororo may be an extension of the endemic Busoga focus. The genetic significance of the high frequency of the heterozygous PGM3/ICD-3 combination (Tait, 1980) in trypanosomes from humans at Tororo, in the absence of the homozygous PGM-2/ICD-2 parent type remains unknown, but such an over-representation of heterozgotes has previously been shown to be indicative of a primarily uniparental method of reproduction (Tibayrenc et al., 1990, 1991). Indeed, a recent study of genetic processes in T.brucei spp from Tororo by Stevens and Welburn (1993) did not support the concept of a randomly mating population of trypanosomes circulating within the epidemic. Whatever the means for genetic change in trypanosomes, the distribution of PGM and ICD isoenzyme patterns appear to provide useful markers for identifying those strains responsible for human sleeping sickness in south-east Uganda. Contingency table analyses of these markers provided support for such a conclusion, and indicated relationships between the distribution of certain PGM and ICD patterns from Busoga and Tororo. Analyses of trypanosome stocks from humans indicated significant associations between, place of origin, ICD or PGM pattern, and strain group. Trypanosomes in the zambezi strain group were isolated significantly more frequently from Busoga than they were from Tororo, while stocks in the busoga strain group were isolated in approximately equally numbers from the two sites; however, the sampling bias indicated that busoga stocks are overrepresented in the Tororo focus. Thus, it is not altogether suprising that the analyses of ICD and PGM patterns also confirmed these findings. For domestic animals ICD pattern appeared most important; trypanosomes with ICD-1, characteristic of the zambezi strain group, were sampled more often from Busoga than from Tororo, while ICD-3 was evenly distributed in the two sites; again, the sample bias indicated a considerable over-representation of ICD-3 in domestic animals from the Tororo focus. In contrast, for the pooled dataset only PGM appeared to be a useful marker. The isoenzyme analysis also detected some non-East African associations. The PGM-2/ICD-2 combination which is characteristic of West African animal trypanosomiasis (bouaflk strain group) and T.b.gambiense (Stevens and Godfrey, 1992) was found in five cattle and a dog in Busoga, and in an isolate from a tsetse fly from Mukono. This may indicate that trypanosome strains of West African origin have become recently established in the Busoga area. However, the combination was isolated once before in Busoga, from a patient in 1977 (Gibson and Gashumba, 1983), and also in an isolate from a patient in south-west Ethiopia (Gibson et al., 1980). Similarly the combination of PGM-2/ICD-1 associated with animals and tsetse has been previously isolated in Kenya and Tanzania (kiboko strain group, Godfrey et al., 1990), but has not before been described from Busoga. Again this result may indicate the recent introduction of 'foreign' strains to Busoga.

112

Alternatively, the detection of these various unusual combinations may simply be the result of possibly the largest ever collection for characterization of trypanosomes from Busoga. The concept of at least two enzymically distinct groups of trypanosomes infecting man and domestic animals in south-east Uganda is strongly supported by the numerical analysis, and the dendrogram places all, but one, zymodemes from man in two discrete clusters. Moreover, as many zymodemes have been previously described (Gibson et al., 1980; Gibson and Gashumba, 1983; Godfrey et al., 1990; Stevens et al., 1992), the clusters are readily matched to the strain groups busoga and zambezi (Gibson et al., 1980). In particular, the majority of trypanosomes in the zarnbezi strain group were isolated from the Busoga area, while the majority of trypanosomes in the busoga strain group were isolated from the Tororo focus; such a result may have serious implications for the spread of the disease. Gibson and Gashumba (1983) identified six different zymodemes among trypanosome stocks isolated from humans in the Busoga area between 1976-1981, including one zymodeme first sampled in 1959. In the current study, zymodemes matched to those identified by Gibson and Gashumba (1983) were seen in both Busoga (Z319) and Tororo (Z17, Z402), thus again indicating that the new focus of infection in Tororo is probably an extension from Busoga. Indeed, this confirms local reports of the spread of the disease and UTRO surveillance records (Enyaru et al., 1992). The enzymic diversity of the trypanosomes isolated from man in the current study (24 zymodemes) was considerably higher than that observed (6 zymodemes) in man in the study by Gibson and Gashumba (1983), even allowing for differences in enzyme system sensitivity. The large number of different zymodemes found in trypanosomes from domestic animals and tsetse also suggests an increase in genetic diversity, which may have important implications for trypanosome population genetics in the region. Other studies have produced comparable results. A broad study of Trypanozoon by Stevens et al. (1992) classified 37/44 stocks, isolated from man and domestic animals in Tororo (Maudlin et al., 1990), as busoga. Again, the distribution of the stocks into zymodemes was highly aggregated, with 33 stocks in just three busoga zymodemes. A recent analysis of this data by Stevens and Welburn (1993) suggests that trypanosome populations circulating in the Tororo epidemic are basically clonal, while the current study showed a high frequency of a heterozygous PGM/ICD combination, apparently in the absence of one homozygous parent type. A taxonomic study of T.b.rhodesiense by Hide et al. (1991) included 17 isolates from Tororo, analysed using RFLP. Their dendrogram divided the stocks into two discrete groups, one comprising human serum resistant animal isolates and human stocks, and the other, human serum sensitive isolates. Serum resistant trypanosomes exhibited relatively homogeneous RFLP patterns which were closely related to stocks previously isolated from south-east Uganda and west Kenya in 1958, 1981 and 1982. This provides further indication that at least some recently isolated Tororo populations have been circulating in the Busoga area since 1958, and that the disease may have spread northwards from the shores of Lake Victoria to Tororo. Such a movement is in agreement with documented evidence (Mbulamberi, 1989). Of the 30 zymodemes identified in domestic animals, nine were also sampled from man. This is consistent with the hypothesis that domestic animals are reservoirs of the human disease in south-east Uganda (Okuna et al., 1986); this may have impor-

113 tant consequences for future sleeping sickness control programmes. Three zymodemes from man were also isolated from tsetse flies, but it is difficult to assess the proportion of flies capable of transmitting human disease (Jordan, 1974), This is particularly true for south-east Uganda (Okoth and Kapaata, 1986) where T.brucei spp infections rates of only 0.14% have been detected (Maudlin et al., 1990). Indeed, in the current study we were able to obtain only one trypanosome isolate from tsetse at Tororo, supporting the report of a low infection rate in the area (Maudlin et al., 1990). Otieno et al. (1990) and Cibulskis et al. (1992) suggest that the transmission of T.brucei spp may be highly localized. In the current study 42% of zymodemes from tsetse were also found in man or domestic animals. The remaining zymodemes may have been contracted while feeding on a wild host, which are difficult to sample accurately on an adequate scale. However, fly bloodmeal analysis by Maudlin et al. (1990), indicates that wild animals appear to play little or no part in the epidemiology of the disease in the Tororo area. The zymodemes found uniquely in tsetse may actually occur in man or domestic animals, but rarely, so that they were not sampled in this study. Alternatively, the zymodemes found only in flies may be highly specific clones (Tibayrenc et al., 1991), or the product of genetic exchange (Jenni et al., 1986). Whatever, overall the high level of genetic diversity in tsetse flies is in agreement with the studies of Gibson and Wellde (1985), Godfrey et al. (1990) and Mihok et al. (1990). In conclusion, this study demonstrates links between trypanosome strain group, geographical location and clinical disease in south-east Uganda. The apparent movement of certain genetic types of trypanosome over a period of ten or more years suggests that the disease has spread from one area to another, while some trypanosomes may have undergone a genetic change. All these aspects require further investigation towards a better understanding of epidemiology, clinical disease and transmission cycles.

Acknowledgements This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases; J.R.S. is supported by the Minist6re Fran~ais des Affaires Etrang~res, France. We thank the Director of UTRO, Uganda for permission to publish this paper, D.H. Smith of the Liverpool School of Tropical Medicine for communicating unpublished clinical information, and D.G. Godfrey and M. Tibayrenc for valuable comments on the manuscript.

References Abaru, D.E. (1985) Sleepingsickness in Busoga, Uganda, 1976-1983. Tropenmed. Parasitol. 36, 72-76. Apted, F.I.C. (1970) Clinical manifestations and diagnosis of sleeping sickness. In: The African Trypanosomiases (ed. H.W. Mulligan), pp. 661-683, George Allen and Unwin, London. Bailey, J.W. and Smith, D.H. (1992) The use of the acridine orange QBC technique in the diagnosis of African trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg. 86, 630.

114 Borst, P., Fase-Fowler, F. and Gibson, W.C. (1981) Quantitation of genetic differences between Trypanosoma brucei garnbiense, rhodesiense and brucei by restriction enzyme analysis of kinetoplast DNA. Mol. Biochem. Parasitol. 3, 117-131. Buyst, H. (1974) The epidemiology, clinical features, treatment and history of sleeping sickness on the northern edge of the Luangwa fly belt. Medical J. Zambia 8, 2-12. Cibulskis, R.E. (1992) Genetic variation in Trypanosoma brucei and the epidemiology of sleeping sickness in the Lambwe Valley, Kenya. Parasitology 104, 99-109. Enyaru, J.C.K., Odiit, M., Gashumba, J.K., Carasco, J.F. and Rwendeire, A.J.J. (1992) Characterization by isoenzyme electrophoresis of Trypanozoon stocks from sleeping sickness endemic areas of southeast Uganda. Bull. WHO 70, 631-636. Enyaru, J.C.K., Allingham, R., Bromidge, T., Kanmogne, G.D., and Carasco, J.F. (1993) The isolation and genetic heterogeneity of Trypanosoma brucei gambiense from north-west Uganda. Acta Trop. In press. Gibson, W.C., Borst, P. and Fase-Fowler, F. (1985) Further analysis of intraspecific variation in Trypanosoma brucei using restriction site polymorphisms in the maxi-circle kinetoplast DNA. Mol. Biochem. Parasitol. 15, 21-36. Gibson, W.C. and Gashumba, J.K. (1983) Isoenzyme characterization of some Trypanozoon stocks from a recent trypanosomiasis epidemic in Uganda. Trans. R. Soc. Trop. Med. Hyg. 77, 114-118. Gibson, W.C., Marshall, T.F. de C and Godfrey, D.G. (1980) Numerical analysis of enzyme polymorphism: a new approach to the epidemiology of trypanosomes of the subgenus Trypanozoon. Advs. Parasitol. 18, 175-246. Gibson, W.C., Mehlitz, D., Lanham, S.M. and Godfrey, D.G. (1978) The identification of Trypanosoma brucei gambiense in Liberian pigs by isoenzymes and by resistance to human plasma. Tropenmed. Parasitol. 29, 335-345. Gibson, W.C. and Wellde, B.T. (1985) Characterization of Trypanozoon stocks from the South Nyanza sleeping sickness focus in Western Kenya. Trans. R. Soc. Trop. Med. Hyg, 79, 671-676. 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. Advs. Parasitol. 29, 1-74. Herbert, W.J. and Lumsden, W.H.R. (1976) Trypanosoma brucei: A rapid "matching" method for estimating the host's parasitaemia. Exp. Parasitol. 40, 427-431. Hide, G., Buchanan, N., Welburn, S., Maudlin, I., Barry, J.D. and Tait, A. (1991) Trypanosoma brucei rhodesiense: Characterisation of stocks from Zambia, Kenya and Uganda using repetitive DNA probes. Exp. Parasitol. 72, 430-439. Hide, G., Cattand, P., Le Ray, D., Barry, J.D. and Tait, A. (1990) The identification of Trypanosoma brucei subspecies using repetitive DNA sequences. Mol. Biochem. Parasitol. 39, 213-226. Jaccard, P. (1908) Nouvelles recherches sur la distribution ftorale. Bull. Soc. Vaudoise Sci. Nat. 44, 223-270. Jenni, L., Marti, S., Schweizer, J., Betschart, B., Lepage, R.W.F., Wells, J.M., Tait, A., Paindavoine, P., Pays, E. and Steinert, M. (1986) Hybrid formation between African trypanosomes during cyclical transmission. Nature 322, 173-175. Jordan, A.M. (1974) Recent developments in the ecology and methods of control of tsetse flies (Glossina spp.) (Dipt., Glossinidae) - a review. Bull. Ent. Res. 63, 361-399. Lanham, S.M. and Godfrey, D.G. (1970) Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 28, 521-534. Lanham, S.M., Grendon, J.M., Miles, M.A., Povoa, M.M. and de Souza, A.A.A. (1981) A comparison of electrophoretic methods for isoenzyme characterization of trypanosomatids, h Standard stocks of Trypanosoma cruzi zymodemes from north-east Brazil. Trans. R. Soc. Trop. Med. Hyg. 75, 742-750. Maudlin, I., Welburn, S.C., Gashumba, J.K., Okuna, N. and Kalunda, M. (1990) The r61e of cattle in the epidemiology of sleeping sickness in Uganda. VII International Congress of Parasitology, Paris. Bull. Soc. franqaise Parasitol. 8, (Suppl. 2), 788. Mbulamberi, D.B. (1989) Possible causes leading to an epidemic outbreak of sleeping sickness. Facts and hypotheses. Ann. Soc. beige Med. Trop. 69, 173-179. Mihok, S., Otieno, LH. and Darji, N. (1990) Population genetics of Trypanosoma brucei and the epidemiology of human sleeping sickness in the Lambwe Valley, Kenya. Parasitology I00, 219-233. Okoth, J.O. and Kapaata, R. (1986) Trypanosome infection rates in Glossinafuscipesfuscipes Newst. in the Busoga sleeping sickness focus, Uganda. Ann. Trop. Med. Parasitol. 80, 459-461. Okuna, N.M., Mayende, J.S.P. and Guloba, A. (1986) Trypanosoma brucei infection in domestic pigs in a sleeping sickness epidemic area of Uganda. Acta Trop. 43, 183-184.

115 Ormerod, W.E. (1961) The epidemic spread of Rhodesian Sleeping Sickness 1908-1960. Trans. R. Soc. Trop. Med. Hyg. 55, 525-538. Ormerod, W.E. (1963) A comparative study of growth and morphology of strains of Trypanosoma rhodesiense. Exp. Parasitol. 13, 374-385. Otieno, L.H., Darji, N. and Onyango, P. (1990) Electrophoretic analysis of Trypanosoma brucei subgroup from cattle, tsetse and patients from Lambwe Valley, Western Kenya. Insect Science and its Application 11,281-287. Rickman, L.R. (1974) Investigations into an outbreak of human trypanosomiasis in the lower Luangwa Valley, Eastern Province, Zambia. East African Med. J. 51,467-487. Robertson, D.H.H. and Baker, J.R. (1958) Human trypanosomiasis in south-east Uganda. 1. A study of the epidemiology and present virulence of the disease. Trans. R. Soc. Trop. Med. Hyg. 52, 337-348. Sokal, R.R. and Michener, C.D. (1958) A statistical method for evaluating systematic relationships. Univ. Kansas Sci. Bull. 38, 1409-1438. Stevens, J.R., Nunes, V.L.B., Lanham, S.M. and Oshiro, E.T. (1989) Isoenzyme characterization of Trypanosoma evansi isolated from capybaras and dogs in Brazil. Acta Trop. 46, 213-222. Stevens, J.R. and Cibulskis, R.E. (1990) Analysing isoenzyme band patterns using similarity coefficients: a personal computer program. Comp. Meth. Prog. Biomed. 33, 205-212. Stevens, J.R. and Godfrey, D.G. (1992) Numerical taxonomy of Trypanozoon based on polymorphisms in a reduced range of enzymes. Parasitology 104, 75-86. Stevens, J.R., Lanham, S.M., Allingham, R. and Gashumba, J.K. (1992) A simplified method for identifying subspecies and strain groups in Trypanozoon by isoenzymes. Ann. Trop. Med. Parasitol. 86, 9-28. Stevens, J.R. and Welburn, S.C. (1993) Genetic processes within an epidemic of sleeping sickness in Uganda. Parasitol. Res., 79, 421-427. Tait, A. (1980) Evidence for diploidy and mating in trypanosomes. Nature 287, 536-538. Tait, A., Barry, J.D., Wink, R., Sanderson, A. and Crowe, J.S. (1985) Enzyme variation in Trypanosoma brucei spp. 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. Proc. Natl. Acad. Sci. USA 87, 2414-2418. Tibayrenc, M., Kjellberg, F., Arnaud, J., Oury, B., Breniere, S.F., Darde, M.-L. and Ayala, F.J. (1991) Are eukaryotic microorganisms clonal or sexual? A population genetics vantage. Proc. Natl. Acad. Sci. USA 88, 5129 5133. Wurapa, F.K., Dukes, P., Njelesani, E.K. and Boatin, B. (1984) A "healthy carrier" of Trypanosoma rhodesiense: a case report. Trans. R. Soc. Trop. Med. Hyg. 78, 349-350.