The subspecific taxonomy of Trypanosoma brucei - Parasite [PDF]

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T h e s u b s p e c if ic t a x o n o m y o f T r y p a n o s o m a b r u c e i J. R. BAKER*

R ésum é

S u m m ary :

Trypanosoma brucei was first seen by David Bruce in 1894, in the blood of a cow in South Africa, and named in his honour in 1899. Trypanosomes seen in the blood of an Englishman in The Gambia in 1901 were named T. gambiense in 1902. Finally, in 1909, trypanosomes from the blood of an Englishman in Zambia

: L a t a x o n o m ie s o u s - s p é c if iq u e d e T r y p a n o s o m a b r u c e i

Trypanosoma brucei a été observé pour la première fois en 1894 par David Bruce, dans le sang d'une vache d'Afrique du Sud, et ainsi nommé en son honneur en 1899. Les trypanosomes observés dans le sang d'un anglais en Gambie en 1901 furent appelés T. gambiense en 1902. Enfin, en 1909,

("Rhodesia") were named T. rhodesiense. Since then there has

les trypanosomes provenant du sang d'un anglais en Zambie

been continuous debate about the interrelationships of these three "species". Studies of the molecular biology of these trypanosomes,

("Rhodésie") furent appelés T. rhodesiense. Depuis, les interrelations entre ces trois "espèces" ont fait l'objet d'un débat continuel. L'étude

mainly analyses of their isoenzymes and deoxyribonucleic acid,

de la biologie moléculaire de ces trypanosomes, principalement

now appear to have shown that T. "rhodesiense" cannot be distin­ guished from T. brucei brucei by any valid and consistent criterion,

l'analyse de leurs isoenzymes et acide désoxyribonucléique, sem­ blent avoir montré que T. “rhodesiense" ne peut être différencié de T.

while T. "gambiense" probably does constitute a valid subspecies of T. brucei. There is still doubt whether populations of T. brucei

gambiense constitue vraisemblablement une sous-espèce de T. bru­

brucei brucei par aucun critère valable et permanent, alors que T.

exchange undoubtedly can occur in this species, its nature and fre­

cei. Il persiste un doute quant au caractère principalement clonal ou sexuel des populations de T. brucei. Bien qu'une mutation génétique

quency are unknown and there is evidence that the population

puisse indubitablement survenir chez cette espèce, sa nature et sa

are predominantly clonal or sexual. W hile some form of genetic

fréquence sont inconnues et il est démontré que la structure démo­

structure of T. brucei is essentially clonal. KEY WORDS :

Trypanosoma brucei.

history, subspecific taxonomy, popula­

MOTS CLÉS : Trypanosoma brucei. histoire, taxonomie sous-spécifique, structure démographique.

tion structure.

BACKGROUND

I

n Novem ber 1894, just over 100 years ago, David Bruce (Fig. 1) (then a captain in the British army and subsequently knighted as Sir David) and his wife Mary arrived in a small village ( ‘a m agistrate’s office and a few mud huts’ : Joubert et al., 1993) cal­ led U bom bo ( O b o n jen i in the local language, accor­ ding to Jo u b e r t e t a l ., 1 9 9 3 ) in K w aZ u lu , N atal Province, South Africa. They had been sent there by the military authorities to try to discover the cause o f a disease know n as n a g a n a w hich was devastating the cattle o f the inhabitants o f the region and the horses used by the administrators o f what was then a British colony. Bruce and his wife established their hom e (and labo­ ratory) in a rectangular building (Fig. 2), built mainly o f m ud o n a fra m e w o rk o f w a ttle ( in te r la c e d branches o f small trees and shrubs) apparently with a

* Royal So ciety o f T ropical M edicine and H ygiene, M anson H ouse, 26, Portland P lace, London, W 1N 4EY , England.

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graphique de T. brucei est essentiellement clonale.

fou n d ation o f sto n es (Jo u b e rt et a l., 1993; C ook, 1994). Bruce started his investigation by examining daily the blood o f a ‘brown cow ’ suffering from n a g a n a . He observed bacteria, which he at first thought w ere pro­ bably the cause o f the disease, but on the sixth day h e r e c o r d e d in h is n o te s th e p r e s e n c e o f ‘H aem ato zo a’ (M acArthur, 1955). H e su bsequ en tly adm itted (B ru c e , 1915) that he originally thought these ‘Haem atozoa’ w ere small filariae, but within a short time (probably before the end o f 1894) Bruce had concluded that the haem atozoa (Fig. 3) w ere the cause o f n a g a n a and also o f ‘tsetse fly disease’ o f humans, w hich until then had b een thought to b e a separate entity (Bruce, 1895, 1896). A few years later th e ‘H a e m a to z o o n ’ w as n am ed b y P lim m er and Bradford (1 8 9 9 ) as T ry p a n o s o m a b r u c ii [sic], Bruce having sent an infected dog to England. The specific name, w hether printed thus due to a typesetter’s error or a lapse on the part o f the authors, was soon cor­ rected to T. b r u c e i (Nabarro, 1907, p. 112 footnote 1, stated that the ch an g e w as m ade by Laveran and Mesnil, but cited no reference).

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Fig. 2. - D avid B r u c e ’s h u t in U b o m b o , 1 8 9 4 -1 8 9 5 (fro m B r u c e , 1915 :

Lancet, ii, 1-6; p h o to g rap h kin dly su p p lie d b y D r G .C . C o o k [se e C ook, 19941).

Fig. 1. - D avid B ru ce (p h otog rap h taken prob ab ly in 1917, and kindly sup plied b y cou rtesy o f th e Administrator, Royal Society o f T rop ical M ed icine and H ygiene).

Fig. 3. - P hotograph o f D r J . E. Dutton (right) w ith, n ext to him , Mr H. Kelly, th e first kn o w n c a se o f hum an trypa­ n o so m ia sis (1 9 0 1 ). O n e o f th e o th er tw o m en is Dr R. M. Forde; th e id en ­ tity o f th e se c o n d is not kn ow n . T he o rig in a l p h o to g ra p h is a n n o ta te d in D utton ’s handw riting ‘T h e first c a se o f try p a n o so m ia s is’, w ith th e n a m e s o f K elly an d D utton. P h o to grap h kindly su p p lied b y co u rtesy o f M iss Patricia Miller, archivist, and th e Photograph ic D epartm ent o f th e Liverpool Sch o o l o f T ropical M edicine.

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T h e s u b s p e c i f i c t a x o n o m y o f Tr y p a n o s o m a

brucei

Fig. 4. - Sk etch es o f ‘H aem atozo a’ in th e b lo o d o f a dog, from Plate V o f B ru ce (1 8 9 5 ). Later, colou red draw ings o f trypan osom es in B ru ce’s reports w ere m ade b y Mrs (later Lady) B ru ce; th ese sk etch es are cruder, and m ay have b e e n drawn by B ru ce him ­ se lf (p h otog rap h kindly sup plied b y courtesy o f Ms M ary G ib so n , b ib lio g ra p h er, and th e V isual Aids D epartm ent o f th e London Sch ool o f H ygiene and T ropical M edicine).

Only two years after the naming o f T. bru cei, in May 1901, a doctor in the British colonial service in The Gambia named R. M. Fordei saw, in the blood o f a 42 years old Englishman ( ‘H. K.’), subsequently identified from the archives o f the Liverpool School o f Tropical Medicine as H. Kelly; see fig. 4), w ho was master o f a government steam boat on the Gambia river, what he d e s c rib e d as ‘sm all w o rm -lik e , e x tre m e ly a ctiv e bodies’ which he first thought w ere filariae. However, later in 1901 J. E. Dutton, o f the Liverpool School of T ro p ic a l M e d icin e , w as v isitin g B a th u rst in T h e Gambia. Forde show ed his patient, and the ‘worm s’ in his blood, to Dutton, w ho recognized them as trypa­ nosom es (Forde, 1902). Dutton (1902) subsequently nam ed the parasite T. g a m b ie n s e . (D utton actually wrote ‘At present then it is im possible to decide defi­ nitely as to the sp e cies, but if on further study it should be found to differ from the other disease pro­ ducing trypanosomes I would suggest that it be called T ry p an osom a g a m b ie n s é (p. 467); although this is a ‘conditional proposal’ in the terms o f the International Code o f Zoological Nomenclature (third edition, 1985, article 15), such proposals made before 1961 do not prevent availability o f the nam e.) On 17 N ovem ber 1909 trypanosom es w ere seen in the b loo d o f a 26 years old Englishm an w ho had b een travelling along the valley o f the Luangwa river in Zambia (then north-east Rhodesia); he was thought to have b eco m e infected there in Septem ber 1 9 0 9 . O n 2 D e c e m b e r h e w as a d m itte d to th e R o y al Southern hospital in Liverpool, under the care o f Dr 1. R obert M ichael Ford e w as b o rn at C loyne, C ounty Cork, Ireland in 1862 and d ied ‘at his h o m e in W orthing [Sussex, England] on March 27 [1942] at th e age o f 8 6 ’ (C.A.H ., 1948).

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Ronald Ross (w ho is now more famous for his colla­ boration with Patrick Manson on the mosquito trans­ mission o f malaria). The case history o f this patient was reported by Ross and Thom pson (1910, 1911); he w as su b jected to a horrifying battery o f attem pted therapies for his condition, including atoxyl, quinine, m ethylene blue, trypsin, amylopsin, succinam ide o f mercury, ‘izal o il’ [presumably a com m ercial house­ hold disinfectant o f that name], trypan red, potassium iodide, ‘v a ccin e s’ prepared from his ow n trypano­ som es grown in rats, and an extract o f rat peritoneal leucocytes. O n one occasion he was injected with 1 x 109 ‘dead’ trypanosom es in rat’s blood; this, the doc­ tors reported, ‘seem ed to cause no harm’ (Ross and Thom pson, 1911). Perhaps not suprisingly, the unfor­ tunate patient died on 29 Ju n e 1910. He was identi­ fied by Ross and Thom pson (1910), and by Stephens and Fantham (1 9 1 0 ), o n ly as ‘W. A .’, a native o f Northum berland in the UK. D uggan (1 9 7 0 ) nam ed him as W. Armstrong, but gave the date o f his infec­ tion as 1908; this seem s to b e a mistake. B efore his death, trypanosom es w ere isolated from W. A.’s blood and studied (in rats) by Drs Stephen and Fantham at the Liverpool S ch o o l o f T ro p ical M edicine. Stephens and Fantham (1 9 1 0 ) noted the virulence o f this strain, and reported the appearance o f posteronuclear trypomastigotes in the rats’ blood w hich (they thought) distinguished it from T. g a m ­ b ie n s e ; they discussed the possibility that it was (i) a ‘variety’ or ‘local race’ o f T. g a m b ien se , (ii) a subspe­ cies o f T. g a m b ie n s e , or (iii) - ‘our ow n view ’ - a new species, T. rh od esien se. It is interesting that, even at the outset, there should have b een som e doubt in the minds o f the describers

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o f T. rh o d e s ie n s e about its validity as a separate spe­ cies. T h ese doubts have survived until the present day, with scientific opinion swaying first one way and then another.

HOW MANY SPECIES OF 'POLYMORPHIC' TRYPANOSOMES IN AFRICA ? There w ere two early schools o f thought. O ne, which might be called the ‘British’ school, concluded that T. b r u c e i and T. r h o d e s ie n s e w ere ind istin gu ish able, though they recognized that not all ‘strains’ o f T. b ru ­ c e i in fecte d h um ans (K in g h o rn and Y o rk e , 1912; Bruce et al., 1913). The opposite view, that the two w ere distinct species, was held by a group o f German w o rk e rs (T a u te , 1 9 1 3 ; K lein e, 1914, 1923; B e c k , 1914), w hose view was supported by the indisputable fact that six isolates o f T. b r u c e i w ere later show n by direct experim ental inoculation not to be infective to hum ans (T a u te and H uber, 1919 , w ho inclu d ed them selves am ong the 131 recipients o f trypanosom einfected blood). Duke (1921) adhered to the ‘British’ view, suggesting that T. ‘r h o d e s ie n s e ’ and T. b r u c e i’ w ere merely ‘bio­ logical races’ o f T. bru cei, and W enyon (1926), in his great m onograph, discussed the d ifferent opinions and concluded that, while T. g a m b ie n s e was a distinct species, ‘ T. r h o d esien se is merely a strain o f T. b ru c ei in m an.’ G radually, ho w ever, the ‘sep aratist’ G erm an view becam e generally accepted. The question seem ed to be settled by the historically fam ous ‘Tinde exp eri­ m ent’. This experim ent, w hich was conducted by a series o f w orkers at the small experim ental station at T in d e in n o rth e rn T a n z a n ia ( th e n T a n g a n y ik a ), consisted o f the serial passage o f a strain o f trypanosom es, isolated in 1936 from a human patient, alter­ nately through G lo ssin a m o rsita n s and sheep, with periodical testing o f its infectivity to humans either by blood inoculation or tsetse fly bite. The experim ent continu ed for 23 years, and the strain, w hich was considered to be T. rh o d esien se, never lost its ability to infect humans (apart from a few sporadic failures, a ttr ib u te d to fa u lts in t e c h n iq u e ) (W ille tt an d Fairbairn, 1955; Ashcroft, 1959). With hindsight, it is easy to regret that the converse experim ent - serially passaging a strain o f T. b ru c ei k now n not to be infective to hum ans, through G. m o rsita n s and sheep - was not conducted simulta­ neously. As pointed out by Rickman (1977), it would have b een interesting to see w hether such a strain w ould have sp o n tan eo u sly acq u ired the ability to infect humans.

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For many years epidem iological evidence had b een building up to suggest that hum an-infective strains o f T. b r u c e i sen su la to existed in wild 'gam e' mammals, and this was conclusively dem onstrated w hen Heisch et al. (1958) isolated such a strain from T rag elap h u s scrip tu s in Kenya. Eight years later, O nyango et al. (1966) isolated a similar strain from a dom estic cow in the same country. However, this was not perceived as undermining the current con cep t o f the distinction o f the two ‘sp ecies’ - T. b r u c e i and T. rh o d esien se. In the book T h e African trypanosom iases’ (Mulligan & Potts, 1970) - in many w ays a sum m ing-up o f the British con tribu tion to trypanosom iasis research in Africa during the then rapidly ending ‘colonial’ era Hoare (1970a) referred to the three species T. bru cei, T. rh o d es ien se and T. g a m b ie n s e , although he quali­ fied this by adding that, ‘from the zoological point o f v iew ’, they could b e classified as su b sp ecies o f T. b r u c e i and in the next chapter o f the sam e b o o k he wrote that T. rh o d e s ie n s e represented ‘merely a viru­ lent race o f T. g a m b ie n s é (see Hoare, 1970b). In 1925 (quoted by Hoare, 1972) and in 1943 Hoare had sug­ gested that T. r h o d e s ie n s e ’ and T. ‘g a m b i e n s e ’ w ere merely races o f T. bru cei. He later (Hoare, 1966) ele­ vated them to subspecies. In 1970 (Hoare, 1970a, b ) his ideas seem have to b e e n in a fluid state, but finally, in his ‘magnum opus’, Hoare (1972) no longer treated T. r h o d e s ie n s e as a distinct su b sp ecies, but merely as one o f two ’nosodem es' o f T. b r u c e i g a m ­ bien se. In 1967, hum an African trypanosom iasis w as newly reported in south-western Ethiopia and, as a result o f investigations there, my colleagu es and I (B a k er et al., 1970; McConnell et al., 1970) concluded that the disease was, indeed, a new occurrence in that area. In discussing its origin, w e considered three possibili­ ties: that it had always b een covertly present; that it had b een new ly introduced from endem ic areas to the south; or that it had arisen from enzootic T. b r u ­ c e i which had ‘recently acquired the ability to infect m an ’. Although w e regarded the last possibility as ‘in te re stin g ’, w e w ere rath er d ism issiv e o f it and concluded that introduction from the south was the m ost likely ex p la n a tio n (th e q u o ta tio n s are from Baker et al., 1970). However, the idea that these trypanosom es need not necessarily b e categorized into rigid divisions began to take root. Hoare (1967) suggested that the T. b r u ­ c e i group, w hich he had redefined (Hoare, 1964) as fo rm in g th e su b g e n u s T r y p a n o z o o n Lü h e, 1 9 0 6 , should b e regarded as ‘com plexes o f morphologically id e n tica l p o p u la tio n s ’ o r, in th e te rm in o lo g y o f Huxley (1963), ‘polytypic sp ecies’. This con cep t was developed, independently, by two Canadian workers

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T h e su b s p e c ific ta x o n o m y o f Tr y p a n o so m a b r vc e i

(Bardsley and Harmsen, 1973) w ho w ere studying the tryp anosom es o f anuran am phibia. T h ese w orkers conclu ded that the trypanosom es o f Anura form ‘a sp ecies co m p lex co m p osed o f a num ber o f clonal aggregates o f varying degrees o f separateness [one or tw o o f which] can be recognized as separate sp e­ c i e s ...’; th ey ad d ed th e cau tio n ary n o te that ‘W e must... be prepared for the possibility o f finding one or m ore central cores o f highly variable, polymorphic species which may have to remain as unresolved spe­ cies co m p lex es in the eyes o f all ex cep t the m ost ardent “splitters’”. This is a co n cep t w hich I think should be borne in mind w hen considering the sub­ genus T ry p a n o z o o n today, uncom fortable though it may be for ardent ‘splitters’. Experimental investigation o f the interrelationships o f the T. b ru c e i com plex was bedevilled for many years by the lack o f any means o f determining the ability, or inability, o f a particular strain to infect hum ans other than its experim ental inoculation into a human volunteer. The situation changed w hen Rickman and Robson (1970) described the ‘blood incubation infectivity test' (B IIT). If trypanosom es o f T. b n ic e i sen su la to w ere incubated under controlled conditions with human blood (or serum), som e strains lost their infectivity for rats while others retained it; and the pheno­ m enon appeared to correlate well with their inability or ability to infect human beings. It was show n by Rifkin (1978) that the trypanocidal factor w as associated with the serum high density lipoproteins (HDL), and later work has show n it to be located in several different HDL populations (Lorenz et al., 1994). Studies by Hajduk and his collaborators (H ag er et a l., 1994; Hajduk et a l., 1994; see also Rowe, 1994) have show n that the lytic activity resides in a m inor subspecies o f HDL. This com ponent may bind to a surface receptor in the flagellar pocket o f T. b n i c e i and su bsequ en tly en ter the p arasite’s lysosomes. The lysosomal m em branes are then disrupted, resulting in autodigestion o f the parasite cell. Why this does not occur in the hum an-infective trypano­ som es is currently being investigated. At first, the B IIT seem ed to support the distinction b e tw e e n T. b r u c e i and T. r h o d e s ie n s e . H ow ever, more detailed studies by Rickman (1977) show ed that different antigenic variants o f a strain initiated by ino­ culating a single trypanosom e into a rat (a cloned strain) could show different BUT responses, sugges­ ting that the property o f infectivity or noninfectivity to humans was not necessarily fixed. This led Rickman (1977) to suggest that hum an-infective trypanosomes could ‘em erge’ am ong a population o f hitherto noninfective (serum sensitive) individuals. Loss o f human infectivity by strains o f T. rh o d e sien se

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during serial non-cyclical passage (i.e., passage by syringe inoculation) through laboratory rodents had long b e e n k now n to o ccu r. W hat w as surprising, how ever, was that Rickman’s work (1977) seem ed to show that the change could occu r in the opposite direction - from not infective to infective; in other w ords, that T. b r u c e i cou ld spontaneously change into T. rh od esien se. This heretical idea w as given som e support by the fact that, in 1975, a m edical student w orking in a laboratory in Edinburgh, Scotland, becam e acciden­ tally infected with a strain o f T. b r u c e i sen su la to w hich had been isolated from a G lossin a p a llid ip e s in south-east Uganda 15 years earlier (Robertson et al., 1980). This strain had b ee n frequently handled in laboratories without special precautions, and no acci­ dental infection had occurred; it was therefore assu­ m ed to b e n o n -in fe c tiv e to h u m ans - alth o u g h , unfortunately, this had never been deliberately tested. A cloned strain had b een prepared from a single try­ panosom e o f the original isolate, and from this 12 dif­ ferent variable antigenic types had b een selected and cryopreserved. A serological study o f the infected stu­ dent show ed that he had b ee n infected with type ETat 10 (ETat = Edinburgh trypanosom e antigenic type) (Herbert et al., 1980), which was found to be the only one o f this series o f 12 which was resistant to human serum in vitro (Van M eirvenne et al., 1976), and therefore, presumably, the only one to b e infec­ tive to humans. Happily, the unfortunate student fully reco v ered after treatm ent w ith suram in. T h e m ost likely e x p la n a tio n o f this ev en t seem s to b e that human infectivity had b een spontaneously developed by ETat 10, possibly by mutation, since the other ele­ ven types (all o f which had originated from the same trypanosom e) w ere rendered non-infective by human serum.

EVIDENCE FROM RECENT MOLECULAR AND GENETIC STUDIES The modern, m olecular approach to a study o f rela­ tionships within the subgenus Try p a n o z o o n ( T b n i ­ c e i sen su la to) originated with the painstaking studies by Godfrey and his co-w orkers o f the isoenzyme pro­ files o f about o n e thou sand trypanosom e p opu la­ tions, as revealed by starch gel and cellulose acetate electrophoresis. The enorm ous amount o f data thus produced was then analysed with the aid o f a com ­ puter to determ ine the degree o f relatedness o f the v a rio u s is o e n z y m ic g ro u p s (te rm e d zy m o d em es; W H O , 1 9 7 8 ). T h is w ork, w h en first rev iew ed by Gibson et al. (1980), indicated that there could b e ‘no d oubt... that T r y p a n o z o o n stocks form a h om oge-

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neous group [and] that T. b ru cei, T. r h o d esien se and T. g a m b ie n s e are too closely related to deserve sepa­ rate species status...’ (G ibson et al., 1980, p. 199). The study also strongly suggested that T. ev a n s i - a ‘spe­ cies’ not transmitted by G lossin a and chiefly associa­ te d w ith c a m e ls - s h o u ld b e in c lu d e d in th is ‘hom ogeneous group’. G ibson et al. (1980) proposed d istin guishing six su bgro u p s, to w hich they gave names without taxonom ic significance. O ne o f these groups ( ‘gam biense’) correlated fairly well with the ‘c la s s ic a l’ T. g a m b i e n s e , but no o n e o f th e o th er groups could be related clearly with the classical ‘ T. r h o d esien sé or ‘ T. b r u c e i. A later, shorter review by G ibson (1986) o f this and other work concluded that there did exist a group o f stocks which corresponded more or less to the classi­ cal concept o f ‘T. g a m b ien se' as infective to humans, o f low virulence to experim ental animal hosts, and generally confined to Central and W est Africa. This group G ibso n (1 9 8 6 ) referred to as ‘group 1 T. b. g a m b ie n s é ; she also recognized a ‘second, less easily defined group w ith greater... h etero g en eity ’ w hich she provisionally referred to as ‘group 2 T. b. g a m ­ b ie n s é . Group 2 had b een isolated only from Burkina Faso and Côte d’Ivoire. Summarizing and extending this work, Godfrey et al. (1 9 9 0 ) and Stevens and G odfrey (1 9 9 2 ) concluded that most or all o f the zymodemes o f T ry p an ozoon , w h en num erically analysed by the construction o f both a dendrogram and a cladogram, fell into three m ajor divisions or pathways, w hich correspond ed , ‘albeit im perfectly’. These divisions did not, however, correspond with the three classical species or subspe­ cies, but w ere separated more on geographical crite­ ria. O ne division was predominantly East African, one was mainly W est African, and the third formed a ‘resi­ dual g ro u p ’ lin ked w ith the Lake V ictoria region. Using the a cc e p te d n o m en clatu re , G od frey e t a l. (1990) concluded that ‘ T. e v a n si and T. b. g a m b ie n s e appeared to be distinct entities.... The status o f T. b. rh o d e s ie n s e and T. b . b r u c e i was particularly uncer­ tain...’. These authors finally concluded that it may be ‘reasonable’ to retain the name T. b. r h o d esie n s e for their W est African strain groups, w hether or not they are infective to humans, and the name T. b. b r u c e i for an essentially W est African strain group, pred om i­ nantly but not exclusively non-infective to humans. These conclusions have b een broadly supported by the work o f other authors. Tait et a l. (1984, 1985), also using isoen zym e electro p h o resis as a to o l to investigate the com plex relationships within the sub­ genus T ry p an oz oo n , concluded that T. b. g a m b ie n s e was a valid subspecies w hich could be distinguished from other stocks o f the subgenus which they refer­

8

red to as ‘ T. b r u c e i (n o n -g a m b ien se )’ (in the 1984 paper). They concluded that the so-called T. b. r h o d e ­ s ie n se w as ‘a set o f variants’ o f T. b. b r u c e i rather than a true subspecies (Tait et a l., 1985). Paindavoine et al. (1986, 1989) exam ined the deoxyri­ bonucleic acid (DNA) o f som e 70-80 populations o f Try p a n o z o o n by means o f digestion with restriction en d o n u cle a ses, h yb rid ization and electro p h o re sis. These authors concluded that all the T. b. g a m b ie n s e stocks w hich they exam ined had a conserved, speci­ fic DNA band pattern and could b e ‘non-am biguously identified’, form ing a single h o m ogen eou s p opu la­ tion. T he oth er stocks they exam in ed , in contrast, yielded highly variable DNA patterns and w ere distri­ buted am ongst several h eterogeneou s groups. T. b. g a m b ien se , they concluded, was therefore a ‘real sub­ species’ w hereas ‘ T. b. rh od esien se' was not, and they suggested that the h eterogeneity indicated that the ‘n on -gam bien se’ trypanosom es w ere evolving m ore rapidly than T. b. g a m b ie n s e itself (Paindavoine et a l ., 1986). In their later paper (Paindavoine et al., 1989), these authors reported that studies using four DNA probes had show n that all 49 T. b. g a m b ie n s e stocks exam ined possessed the same com bination o f alleles and w ere hom ozygous for the four m arkers used. This study supported the view that the ‘n on -gam ­ b ien se’ stocks w ere diploid and could undergo g en e­ tic exchange (see below ). T. b. g a m b ie n s e , however, they thought w as g en etically isolated and did not exch an ge g enetic m aterial w ith other stocks o f the subgenus T r y p a n o z o o n . This study also supported the claim by Dero et al. (1987) that the genom e o f T. b. g a m b i e n s e c o n ta in e d o n ly a b o u t 70 % o f th e amount o f DNA in the genom e o f the non-g a m b ie n s e stocks, and only about 50% o f the latter’s variable antigen gene repertoire.

CLONAL OR SEXUAL POPULATION STRUC­ TURES ?

I

n spite o f sporadic publications claiming to have d etected sex u a l rep ro d u ctio n am on g tryp an o ­ som es (review ed by Hoare, 1972, pp. 48-51), it was generally accepted until about 15 years ago that genetic exchange did not occur am ong trypanosom es o f the subgenus T ry p a n o z o o n (nor, indeed, am ong any other groups). However, in 1980 Tait again raised the possibility o f genetic exchange occurring, suppor­ ting his claim with evidence o f apparent hybrid for­ mation detected by isoenzym e electrophoresis. T h is w o rk h as b e e n c o n s id e ra b ly e x te n d e d and confirm ed by several w orkers (for exam ple, Jen n i et a l., 1986; Sch w eitzer et a l., 1988; Sternberg e t a l.,

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T h e su b s p e c ific ta x o n o m y o f Tr yp a n o s o m a brucfj

1988, 1989; Gibson, 1989; Paindavoine et a l., 1989; P earson & Jen n i, 1989). It now seem s indisputable that som e form o f genetic exchange can occur during that part o f the life cycle which takes place within G lo s sin a , at som e stage b efo re the p rod u ction o f m etacyclic trypom astigotes. T he details o f the pro­ cess, however, are unclear, and - perhaps suprisingly - no confirmatory cytological evidence o f the process has yet b een obtained. It is also clear that this genetic exch ange is not an obligatory process and, although it may b e frequent b etw een certain stocks (Stern berg et a l., 1989), its occurrence is by no m eans universal. It has b een sug­ gested by Pearson & Jen n i (1989) that som e system o f mating types may be involved, as is well know n to be the case with P a ra m ec iu m . The DNA o f the kineto p la st a p p e a rs n o t to tak e part in this p ro c e ss; Sternberg et al. (1988) show ed that inheritance o f the kinetoplast was uniparental. T h e re a lso se e m s little d o u b t that try p a n o so m es ( T ry p a n o z o o n ) are diploid throughout most o f their life cycle. This was first suggested by Tait (1980), and confirm ed by G ibson et al. (1985), Paindavoine et al. (1989) and Pearson & Jen n i (1989). How ever, Cibulskis (1988), w hile agreeing that the ‘range o f genotypes in T. b ru c ei is most readily explai­ ned by genetic exchange’, rightly pointed out that the frequency o f its occurrence was (and is) not known; it ‘may not occur sufficiently frequently, or in such a w ay as to b re a k up a s s o c ia tio n s b e tw e e n l o c i’. Cibulskis (1988) concluded that ‘it is not clear that sex exerts any control over the relative proportions o f genotypes at individual lo c i.’ This con clu sion is in accord with the views o f Tibayrenc and his co-w orkers (Tibayrenc et al., 1990, 1991; Truc & Tibayrenc, 1993; Mathieu-Daude & Tibayrenc, 1994) who, from a study o f population genetics, concluded that trypano­ som es (and many other parasitic protists) have an essentially clonal population structure, rather than one which is based on sexual reproduction; their analysis has shown that segregation and recombination, which are necessary con sequ ences o f sexual reproduction, are rare or absent in natural populations o f the orga­ nisms concerned. This does not exclude the possibi­ lity that sex u al p ro c e sse s m ay o ccu r, bu t m erely indicates that they may occur infrequently and are not generally o f genetic significance in these populations (T ib a y re n c e t a l ., 1 9 9 0 , 1 9 9 1 ; M ath ieu -D au d e & Tibayrenc, 1994; Mathieu-Daude et al., 1994). A consequence o f this view is that the clones, many o f which appear to be stable over space and time, are the basic taxonom ic unit rather than the conventional Linnean species and subspecies. The three ‘subspe­ cies’ o f T. b r u c e i w ere regarded by Tibayrenc et al.

Parasite, 1995, 2, 3 -12

(1990) m erely as ‘pathotypes’, the species being com ­ p o sed o f ‘n um erous clo n es, som e o f w h ich have b eco m e specialized to hum an h o sts’. Th e T. g a m b ie n s e group 1 o f G ib so n (1 9 8 6 ) is a ‘g en etically h o m o gen eo u s... su ccessful, ubiquitous hum an-host c lo n e ’ (T ib a y re n c e t a l ., 1 9 9 0 ). In a la ter p a p e r (M athieu-Daude and Tibayrenc, 1994) this group o f w orkers used the ‘d em e’ terminology introduced into the trypanosom iasis literature by Hoare (1 955) and referred to the so-called T. b. b r u c e i and T. b. rh o d es i e n s e as n o s o d e m e s ’ rath er th an a ctu al g e n e tic clades; T. b. g a m b ie n s e group 1 (Gibson, 1986) was regarded as a ‘group’ o f clones [not, now, a single clone] consisting o f m ost o f the stocks o f hum aninfective trypanosom es from Central and W est Africa. In another paper, Mathieu-Daude et al. (1994) sho­ w ed that a group o f 12 stocks o f human-infective try­ panosom es from Central and W est Africa w ere closely related by the use o f a specific kinetoplast deoxyribo­ nucleic acid probe, and they identified this group as G ibson’s (1986) group 1 T. b. g a m b ien se. Truc and Tibayrenc (1993) also equated one group of zym odem es, among the 23 w hich they identified in 55 stocks o f T. b r u c e i s e n s u la t o isolated in W est A frica, w ith th e ‘c la s s ic a l’ T. b. g a m b i e n s e . T h e various zymodemes, they suggested, w ere equivalent to ‘natural c lo n e s (o r a fam ily o f c lo se ly related clones), stable in space and tim e.’

CONCLUSIONS

T

he inescapable conclusion from the foregoing brief review seems to me to be the fact that it is futile to attempt to maintain the distinction betw een the erstwhile subspecies (or species) T. b. b ru c ei and T. b. rh od esien se. These organisms must be regarded as a collection o f populations (whether clonally or sexually derived), all fairly closely related Mathieu-Daude & Tibayrenc (1994) pointed out that the total genetic variability detected in a study o f 18 isoenzyme loci amongst 78 stocks o f T. b ru c ei sen su lato was less than that seen in the ‘single’ species T. c r u z i - some o f w hich had developed the ability to resist destruction by HDL in human blood; this distinc­ tion, though often durable for quite long periods, can­ not be thought o f as immutable. I am not in favour of the proposal by Godfrey et al. (1990) to redefine T. b. b ru c ei and T. b. rh o d esien se in terms other than their infectivity (o r non-infectivity) to human hosts. This course o f action would, I think, be confusing since the two subspecific names have for so long been used to denote precisely that characteristic.

The situation is different with T. b. g a m b ien se . All the recent w ork discussed above has show n that there

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9

J . R. B A K E R

e x ists a c o re o f ‘ty p ic a l’ h u m a n -in fe ctiv e T ry p a n o z o o n stocks producing chronic human African try­ panosom iasis, based on (but not restricted to) W est Africa, w h ich can fairly certainly b e distinguished from other stocks o f the T. b r u c e i com plex. The argu­ ment has thus, in a sense, swung around a full circle : T. rh o d e s ie n s e is not a valid species, or even subspe­ cies, not b ecau se it is indistinguishable from T. b. g a m bien se, as the early British workers thought, but b e c a u s e it is m erely a g ro u p o f zy m o d em es (o r clon es) o f T. b. bru cei.

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J. E. Preliminary note upon a trypanosome occur­ ring in the blood of man. Thompson Yates Laboratories Reports, 1902, 4, 455-468.

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R.M. Some clinical notes on a European patient in whose blood a Trypanosoma was observed. Jou rn a l o f Tropical Medicine, 1902, 5, 261-263.

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W.C. Will the real T ry p an osom a b. g a m b ie n s e please stand up ? Parasitology Today, 1986, 2, 255-257.

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Parasite, 1995, 2, 3-12