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An attempt to isolate Trypanosoma rhodesiense from wild animals
276 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 52. No. 3. May, 1958.
AN ATTEMPT
TO ISOLATE
TRYPANOSOMA
RHODESIENSE FROM WILD
ANIMALS BY
M. T. A S H C R O F T *
(From Tinde Laboratory, East African Trypanosomiasis Organisation, Tanganyika Territory) Although there is evidence that wild animals m a y act as a reservoir of h u m a n sleeping sickness due to Trypanosoma rhodesiense, this trypanosome has never been isolated f r o m them. This p a p e r records an investigation of which the main purpose was an attempt to do this by isolating strains of polymorphic trypanosomes f r o m wild animals in two areas of Tanganyika in which T. rhodesiense sleeping sickness is endemic, and by testing the infectivity of the strains to man. METHOD
The animals were shot by Mr. W. A. Hilton, and about 0.5 c.c. of blood, taken from the cut throat, was immediately inoculated into each of two white rats, which were kept in fly-proof boxes. Thin and thick blood films were prepared and stained with Giemsa. Each slide was later examined for trypanosomes for 20 minutes by two observers. Fresh films of the blood of the subinoculated rats were examined twice weekly for 90 days for evidence of infection. The polymorphic strain which was isolated was maintained in rats until it was convenient to test its infectivity to man on volunteers. RESULTS
1) Northern Province of Tanganyika ; Mbulu District : Babati area Sleeping sickness occurs in the B a b a t i - D u d a m e r a - M a g u g u area, close to the Great N o r t h Road, 110 to 75 miles south of Arusha. T h e disease was first diagnosed in this region in 1944 ; in the first 3 years of the epidemic a total of 458 cases were found, but the numbers have diminished and in 1953, 1954, and 1955, 50, 41 and 32 cases were diagnosed respectively (APTED, 1955). T h e disease is acquired in a Glossina morsitans area close to Babati and at the foot of the Rift Wall ; also further north in bush infested with G. swynnertoni, where the W a m b u g w e h u n t and collect firewood and honey. I t is difficult to attribute the continuing low endemicity to m a n - f l y - m a n spread because periods of some months occur in which no new cases are diagnosed. JACKSON(1955) described how one of his fly-boys was infected at Daga-Iloi, near Babati ; as there was neither settlement in, nor right of way through, Daga-Iloi, he thought it was unlikely that the tsetse, which had bitten the fl3~-boy, had acquired its infection f r o m another h u m a n being. * I wish to thank Mr. W. A. Hilton for his skilled hunting, and members of the staff of Tinde Laboratory for invaluable assistance.
M, T. ASHCROFT
277
A l l t h e a n i m a l s w e r e s h o t in t h e m o r e n o r t h e r l y G. swynnertoni area f r o m J u l y to S e p t e m b e r , 1956. T h e i n c i d e n c e of t r y p a n o s o m i a s i s w h i c h was f o u n d in t h e s e a n i m a l s is r e c o r d e d in T a b l e I. TABLE I. Results of examination of wild animals for trypanosomes near Babati. No. found to be infected with T. brucei
7?. congolense
T. vivax
I
NO.
Species of animal
examined
By blood fihn
By rat inoc.
By blood film
By rat inoc.
By blood film
Giraffe
Giraffa camelopardalis Ostrich
Struthio camelus Oryx
Oryx beisa Hunting dog
Lycaon pictus Kirk's dikdik
1
Rhyncotragus kirki Klipspringer
1
Oreotragus oreotragus Steinbuck
1
Rhaphicerus campestris Wildebeest
2
Gorgon taurinus Bohor reedbuck
2
Redunca redunca 2
Warthog
Phacochoerus aethiopicus Grant's gazelle
5
GazeUa granti Thomson's gazelle
5
Gazella thomsoni Coke's hartebeest
7
Alcelaphus cokei 7
Zebra
Equus burchelli Impala
10
Aepyceros melampus Total N.B.
47
--
1
T h e two infected Coke's hartebeest were different animals.
4
1
278
A T T E M P T TO ISOLATE
Trypanosoma rhodesiense
FROM W I L D A N I M A L S
One strain of polymorphic trypanosomes was isolated from a fully grown female hartebeest (Alcelaphus cokei) and was maintained in rats by four syringe passages ; a mixture of rat blood and citrate saline, containing 4,000,000 and 6,000,000 trypanosomes was then inoculated subcutaneously into the arms of two African volunteers. These men remained well, with no fever and no local reaction ; blood smears were negative and rats subinoculated with their blood were not infected. It was concluded therefore that the strain was "2". brucei
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,~
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AREAS
MENTIONED
IN THE TEXT
SH7 "~"
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UFIPA
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~i
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t~ ),
~
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Map of Tanganyika showing some of the places mentioned in the text.
because it did not infect these men. After 28 days of observation, the volunteers were treated with a course of antrypol as a routine precautionary measure. 2) Western Province of Tanganyika ; Tabora District : Ugalla river area Cases of sleeping sickness originate annually from near the Ugalla river although the fishermen and the honey hunters, who are the only inhabitants, are said to be absent during the rains, which last for 6 months of the year. JACKSON (1955) described 25 cases of the disease occurring in fly-boys stationed in remote outposts in this area between 1935 and 1939,
M. T. ASHCROFT
279
and he concluded that this was strong evidence that the game was acting as a reservoir. T h e Ugalla river is part of the Malagarasi river system which lies in the Western Province and drains into Lake Tanganyika. Over half the cases of T. rhodesieme sleeping sickness diagnosed in Africa in 1953, 1954, and 1955, were contracted in the vast, sparsely populated area drained by this river system, which is covered, for the most part, with woodland, inhabited by many wild animals and infested with G. morsitans. T h e incidence of sleeping sickness in the Western Province in which the area mainly lies for these 3 years was 538, 895, and 636, cases respectively (APTED, 1955). T h e animals were shot, during September and October, 1956, near the north bank of the Ugalla river, a few miles west of the Ugalla River Game Controlled Area. G. morsitans is the only species of tsetse present. T h e incidence of trypanosomiasis found in the animals is recorded in Table II. No strains of polymorphic trypanosomes were isolated. COMMENT Faihtre to isolate T. rhodesiense
T h e only strain of polymorphic trypanosomes to be isolated proved to be T. brucei. TABLE II.
Results of examination of wild animals for trypanosomes near the Ugalla River. No. found to be infected with T. congolense
T. brucei No.
Species of animal Civet cat
examined
By blood film
By rat inoc.
By blood film
By rat inoc.
T. vivax
By blood film
1
Viverra civetta
Warthog
2
1
Phacochoerus aethiopicus
Oribi
4
Ourebia ourebi
Topi
5
Damaliscus korrigum
Roan
5
Egocerus equinus
Lichtenstein's hartebeest
5
dlcelaphus lichtensteini
Southern reedbuck
4
--
2
Redunca arundinum
Bohor reedbuck
1
1
Redunca redunca
Total
27
l
3
1
N.B. Only two southern reedbuck were found to be infected. One of these animals was infected with both T. vivax and T. congolense.
280
ATTEMPT TO ISOLATE
Trypanosoma rhodesiense FROM
W I L D ANIMALS
This experiment was the first deliberate attempt which has yet been made to infect man with strains from wild animals living in areas in which sleeping sickness is known to be endemic. A number of experiments have been carried out, all with negative results, to test the infectivity to man of strains derived from animals, e.g. TAUTE (1913), but as these strains were obtained in areas in which sleeping sickness did not occur, their failure to infect man was not surprising. Although the investigation failed to demonstrate the existence of T. rhodesiense in wild animals, little light is thrown on the problem as to the part animals play in the epidemiology of the disease, because only one strain of polymorphic trypanosomes was isolated. There is, however, strong evidence both experimental and epidemiological, for the belief that animals do act, in certain circumstances, as a reservoir. The experimental evidence is particularly convincing. At Tinde laboratory, a strain of T. rhodesiense has been transmitted through sheep by G. morsitans since 1934, and the strain was also passed through various species of antelope for 10 years and monkeys for 6 years (FAIRBAIRN and BUI~TT, 1946 ; WILLETT and FAIRBAIRN, 1955). Human volunteers were infected by single G. morsitans which had fed on infected sheep, monkeys, Thomson's gazelle, impala, dikdik, steinbuck, bushbuck, reedbuck, and bushpig. The strain has retained its infectivity to man throughout the experiment and a volunteer was infected in December, 1957, after the strain had been transmitted through sheep for over 23 years. Various other animals, including eland, duiker and warthog, as well as the species mentioned above, were infected with T. rhodesiense. The infections died out after varying lengths of time but successful fly transmissions were obtained after quite long periods from some animals. For instance, a successflfl fly transmission was obtained 10 months after the initial infection of a reedbuck, and 140 days after the infection of an impala. DuKE (1935, 1937) was able to transmit I". rhodesiense from a hyaena, which had been infected 569 days, and a bushbuck, which had been infected about 600 days, to monkeys and thence to man. Considering these results, it would be very surprising if a T. rhodesiense infection of a wild animal, in natural conditions, never exists. G. pallidipes have been found to carry T. rhodesiense on three occasions (McKIcHAN, 1944 ; E.A.T.R.O. Annual Reports, 1956, and in press) ; it is known that a favoured source of food of G. pallidipes is bushbuck (WEITZ and GLASGOW, 1956), and DUKE (1935, 1937) showed that bushbuck can be infected with T. rhodesiense and carry this trypanosome in a form transmissible to tsetse and infective to man for a long period ; it is therefore highly likely that some bushbuck, in areas where sleeping sickness is carried by G. pallidipes, are infected with T. rhodesiense. Thomson's gazelle, reedbuck and duiker have been infected with strains of T. brucei and the course of the infections was similar to that of T. rhodesiense in the same species (BURTT, pers. commun.). This similarity in infectivity of the two trypanosomes is found in all animals which have been experimentally infected except man ; and as wild animals are a reservoir of T. brucei it is to be expected that they also act as a reservoir of T. rhodesiense. Although the isolation of T. rhodesieme from a wild animal by an investigation such as is reported in this paper would be proof of the existence of naturally infected animals, it would give little information about the importance of the animal reservoir. The importance of the reservoir can be estimated only by epidemiological studies, from which it is extremely difficult to reach incontrovertible conclusions. In order to prove that a man who had contracted sleeping sickness had not acquired the infection from a tsetse which had previously fed on another infected man, that is, to prove that the infection of the tsetse was derived from
M. T. ASRCROFT
281
an animal, one must be certain that no other infected men in the previous 6 m o n t h s - - w h i c h is about the maximum life span of the tsetse - - had passed through the areas in which the man might have contracted the disease. In the conditions of the African bush, with the wandering hunters and fishermen, and considering the sometimes chronic nature of the disease, it is almost impossible to be certain that these conditions are fulfilled. JACKSON (1955), however, described 25 cases of sleeping sickness in his fly-boys living in remote and little visited camps ; he concluded that this was strong evidence that the game was acting as a reservoir because he considered that the chances of the tsetse's transmitting the disease from man to man, in the conditions at these camps, must have been very small indeed. If it is difficult to prove from epidemiological data that wild animals do act as a reservoir of T. rhodesiense, it is even more diflqcult to prove that they do not; such a proof would require certain knowledge that the tsetse vectors in an area in which the disease was present were not feeding on any animals capable of being infected with T. rhodesiense ; this condition is hardly possible as the experiments quoted above have shown that a large number of species can be infected with this trypanosome. In the absence, therefore, of incontrovertible epidemiological proof that the wild animals do, or do not, act as a reservoir of sleeping sickness due to T. rhodesiense, we can only consider the likelihood that they do so, bearing in mind that experiments have shown that many species on which the tsetse feed can be infected. T h e large n u m b e r of cases of the disease which are contracted in the fly belt in the Western Province of Tanganyika, where men are scarce but wild animals are numerous and where the tsetse vector, G. morsitans, is a zoophilic species, suggests that not only is there an animal reservoir but that the part it plays is an important one. In other areas where the disease occurs, the importance of such a reservoir may be less. T h e similarities between T. brucei and T. rhodesiense are so striking, especially their infectivity to all animals except man, that I believe that both are primarily parasites of wild animals with the important and stable difference that T. rhodesiense can also infect man.
The incidence of trypanosomiasis T h e proportion of animals found to be infected with all species of trypanosomes, 10 in 74, (13.5 per cent.) was lower than that found in most previous investigations. KINGHORNand YORKE (1913) found a 22 per cent. incidence in 251 animals in N o r t h e r n Rhodesia ; they used as diagnostic methods the examination of blood films and subinoculations into rats and monkeys. BRUCEet al. (1914) found a 31.7 per cent. incidence in 180 animals in Nyasaland ; diagnosis was by blood films and subinoculations into goats, monkeys and dogs. BUCHANAN (1929) found a 21 per cent. incidence in 150 animals in the Ufipa district of Tanganyika ; diagnosis was by the examination of blood films only. VANDeReLANK (1947) found a 17.5 per cent. incidence in 378 animals from various areas of the Lake Province of Tanganyika : diagnosis was from blood films only. Although the results may represent to some extent true differences between areas, a n u m b e r of factors may influence the incidence found ; these are discussed below. Methods of diagnosis. Neither the examination of blood films nor the subinoculations into rats is a completely satisfactory method. In the present investigation a single trypanosome was seen on some slides, suggesting that more prolonged examination might give a greater n u m b e r of positive findings. Rat subinoculation is of limited value because T. vivax and some strains of T. congolense are unable to infect rats. T h e limitations of both methods
282
ATTEMPT TO ISOLATE
Trypanosoma rhodesiense FROM
WILD ANIMALS
are brought out by the fact that, in the present experiment, when an infection was diagnosed from a blood slide, rats were not infected, and vice versa. There is no satisfactory method available for the diagnosis of cryptic trypanosomiasis and it is not impossible that nearly every animal carries trypanosomes, albeit in very small numbers. The species of animal. An analysis of the results of KINGHORN and YORKE (1913), BRUCE et al. (1914), BUCI~ANAN (1929) and VANDERPLANI~ (1947), show that the greatest incidence of trypanosomiasis has been found in waterbuck, bushbuck, reedbuck, kudu, giraffe and eland ; although these six species comprised over 29 per cent. of all the animals examined, they represented 63 per cent. of those found to be infected with trypanosomiasis. Few anim~als of these species were examined in the present investigation and this may be partially responsible for the low incidence found. The season. VANDERPLANK(1947) found a seasonal variation in the number of animals detected with trypanosomiasis, the highest incidence being during the rains. T h e experiments described here took place at the end of the dry season. SUMMARY
1) An attempt was made to isolate strains of polymorphic trypanosomes from wild animals in two areas of endemic T. rhodesiense sleeping sickness in Tanganyika by the subinoculation of blood into rats. One strain of polymorphic trypanosomes was isolated but it did not infect two volunteers. Direct proof of the presence of T. rhodesiense in wild animals is, therefore, still lacking• 2) Blood films from the animals were also examined. Of a total of 74 animals, 10 were found to be infected with T. brucei, T. congolense or T. vivax. The probability is discussed that the true incidence is higher because cryptic infections can be missed. REFERENCES APTED, F. I. C. (1956). Tanganyika Medical Department. Sleeping Sickness Service Annual Report, 1955. BRUCE, D., HARVEY,D., HAMERTON,A. E., DAVEY,J. B. gg LADYBRUCE(1914). Rep. Sleeping Sickness Comm., 15, 16. BUCHANAN,J. C. R. (1929). E. Aft. reed. J., 4, 111. DUNE, H. L. (1935). Parasitology, 25, 171. - (1937). Ibid., 29, 12. East African Trypanosomiasis Research Organisation (1956). Annual Report, 1955-56. • Annual Report, 1956-57 (in press). FAIRBAIRN,H. gg BURTT, E. (1946). Ann. trop. Med. Parasit., 40, 270. JACKSON,C. H. N. (1955). Trans. R. trop. Med. Hyg., 49, 582. KINGHORN,A. 6c YORKE,W. (1913). Ann. trop. Med. Parasit., 7, 184. McKIcI-IAN, J. W. (1944). Trans. R. Soe. trop. Med. Hyg., 38, 49. TAUTE, M. (1913). Abstract in Trop. Dis. Bull., 2, 348. VANDERPLANK,F. L• (1947). Ann. trop. Med. Parasit., 41, 365. WEITZ, B. • GLASGOW,J. P. (1956). Trans. R. Soc. trop. Med. Hyg., 50, 593. WILLETT, K. C. & FAIRBAIRN,It. (1955). Ann. trop. Med. Parasit., 49, 278.
AN ATTEMPT
TO ISOLATE
TRYPANOSOMA
RHODESIENSE FROM WILD
ANIMALS BY
M. T. A S H C R O F T *
(From Tinde Laboratory, East African Trypanosomiasis Organisation, Tanganyika Territory) Although there is evidence that wild animals m a y act as a reservoir of h u m a n sleeping sickness due to Trypanosoma rhodesiense, this trypanosome has never been isolated f r o m them. This p a p e r records an investigation of which the main purpose was an attempt to do this by isolating strains of polymorphic trypanosomes f r o m wild animals in two areas of Tanganyika in which T. rhodesiense sleeping sickness is endemic, and by testing the infectivity of the strains to man. METHOD
The animals were shot by Mr. W. A. Hilton, and about 0.5 c.c. of blood, taken from the cut throat, was immediately inoculated into each of two white rats, which were kept in fly-proof boxes. Thin and thick blood films were prepared and stained with Giemsa. Each slide was later examined for trypanosomes for 20 minutes by two observers. Fresh films of the blood of the subinoculated rats were examined twice weekly for 90 days for evidence of infection. The polymorphic strain which was isolated was maintained in rats until it was convenient to test its infectivity to man on volunteers. RESULTS
1) Northern Province of Tanganyika ; Mbulu District : Babati area Sleeping sickness occurs in the B a b a t i - D u d a m e r a - M a g u g u area, close to the Great N o r t h Road, 110 to 75 miles south of Arusha. T h e disease was first diagnosed in this region in 1944 ; in the first 3 years of the epidemic a total of 458 cases were found, but the numbers have diminished and in 1953, 1954, and 1955, 50, 41 and 32 cases were diagnosed respectively (APTED, 1955). T h e disease is acquired in a Glossina morsitans area close to Babati and at the foot of the Rift Wall ; also further north in bush infested with G. swynnertoni, where the W a m b u g w e h u n t and collect firewood and honey. I t is difficult to attribute the continuing low endemicity to m a n - f l y - m a n spread because periods of some months occur in which no new cases are diagnosed. JACKSON(1955) described how one of his fly-boys was infected at Daga-Iloi, near Babati ; as there was neither settlement in, nor right of way through, Daga-Iloi, he thought it was unlikely that the tsetse, which had bitten the fl3~-boy, had acquired its infection f r o m another h u m a n being. * I wish to thank Mr. W. A. Hilton for his skilled hunting, and members of the staff of Tinde Laboratory for invaluable assistance.
M, T. ASHCROFT
277
A l l t h e a n i m a l s w e r e s h o t in t h e m o r e n o r t h e r l y G. swynnertoni area f r o m J u l y to S e p t e m b e r , 1956. T h e i n c i d e n c e of t r y p a n o s o m i a s i s w h i c h was f o u n d in t h e s e a n i m a l s is r e c o r d e d in T a b l e I. TABLE I. Results of examination of wild animals for trypanosomes near Babati. No. found to be infected with T. brucei
7?. congolense
T. vivax
I
NO.
Species of animal
examined
By blood fihn
By rat inoc.
By blood film
By rat inoc.
By blood film
Giraffe
Giraffa camelopardalis Ostrich
Struthio camelus Oryx
Oryx beisa Hunting dog
Lycaon pictus Kirk's dikdik
1
Rhyncotragus kirki Klipspringer
1
Oreotragus oreotragus Steinbuck
1
Rhaphicerus campestris Wildebeest
2
Gorgon taurinus Bohor reedbuck
2
Redunca redunca 2
Warthog
Phacochoerus aethiopicus Grant's gazelle
5
GazeUa granti Thomson's gazelle
5
Gazella thomsoni Coke's hartebeest
7
Alcelaphus cokei 7
Zebra
Equus burchelli Impala
10
Aepyceros melampus Total N.B.
47
--
1
T h e two infected Coke's hartebeest were different animals.
4
1
278
A T T E M P T TO ISOLATE
Trypanosoma rhodesiense
FROM W I L D A N I M A L S
One strain of polymorphic trypanosomes was isolated from a fully grown female hartebeest (Alcelaphus cokei) and was maintained in rats by four syringe passages ; a mixture of rat blood and citrate saline, containing 4,000,000 and 6,000,000 trypanosomes was then inoculated subcutaneously into the arms of two African volunteers. These men remained well, with no fever and no local reaction ; blood smears were negative and rats subinoculated with their blood were not infected. It was concluded therefore that the strain was "2". brucei
6>
LAKE $
MAP SHOWING
V ICTORIAt'~'-~. \
"~" "\,..
~ ~
7,f r
YANGA
,~
/
t
AREAS
MENTIONED
IN THE TEXT
SH7 "~"
,/ ,,} J
"ERN
UFIPA
Z
~i
z
o
Are,s wheregame was • shot
t~ ),
~
f"~_.,.~"~"~"
-
~7..--'f~',~.__...~ -
/
/
Map of Tanganyika showing some of the places mentioned in the text.
because it did not infect these men. After 28 days of observation, the volunteers were treated with a course of antrypol as a routine precautionary measure. 2) Western Province of Tanganyika ; Tabora District : Ugalla river area Cases of sleeping sickness originate annually from near the Ugalla river although the fishermen and the honey hunters, who are the only inhabitants, are said to be absent during the rains, which last for 6 months of the year. JACKSON (1955) described 25 cases of the disease occurring in fly-boys stationed in remote outposts in this area between 1935 and 1939,
M. T. ASHCROFT
279
and he concluded that this was strong evidence that the game was acting as a reservoir. T h e Ugalla river is part of the Malagarasi river system which lies in the Western Province and drains into Lake Tanganyika. Over half the cases of T. rhodesieme sleeping sickness diagnosed in Africa in 1953, 1954, and 1955, were contracted in the vast, sparsely populated area drained by this river system, which is covered, for the most part, with woodland, inhabited by many wild animals and infested with G. morsitans. T h e incidence of sleeping sickness in the Western Province in which the area mainly lies for these 3 years was 538, 895, and 636, cases respectively (APTED, 1955). T h e animals were shot, during September and October, 1956, near the north bank of the Ugalla river, a few miles west of the Ugalla River Game Controlled Area. G. morsitans is the only species of tsetse present. T h e incidence of trypanosomiasis found in the animals is recorded in Table II. No strains of polymorphic trypanosomes were isolated. COMMENT Faihtre to isolate T. rhodesiense
T h e only strain of polymorphic trypanosomes to be isolated proved to be T. brucei. TABLE II.
Results of examination of wild animals for trypanosomes near the Ugalla River. No. found to be infected with T. congolense
T. brucei No.
Species of animal Civet cat
examined
By blood film
By rat inoc.
By blood film
By rat inoc.
T. vivax
By blood film
1
Viverra civetta
Warthog
2
1
Phacochoerus aethiopicus
Oribi
4
Ourebia ourebi
Topi
5
Damaliscus korrigum
Roan
5
Egocerus equinus
Lichtenstein's hartebeest
5
dlcelaphus lichtensteini
Southern reedbuck
4
--
2
Redunca arundinum
Bohor reedbuck
1
1
Redunca redunca
Total
27
l
3
1
N.B. Only two southern reedbuck were found to be infected. One of these animals was infected with both T. vivax and T. congolense.
280
ATTEMPT TO ISOLATE
Trypanosoma rhodesiense FROM
W I L D ANIMALS
This experiment was the first deliberate attempt which has yet been made to infect man with strains from wild animals living in areas in which sleeping sickness is known to be endemic. A number of experiments have been carried out, all with negative results, to test the infectivity to man of strains derived from animals, e.g. TAUTE (1913), but as these strains were obtained in areas in which sleeping sickness did not occur, their failure to infect man was not surprising. Although the investigation failed to demonstrate the existence of T. rhodesiense in wild animals, little light is thrown on the problem as to the part animals play in the epidemiology of the disease, because only one strain of polymorphic trypanosomes was isolated. There is, however, strong evidence both experimental and epidemiological, for the belief that animals do act, in certain circumstances, as a reservoir. The experimental evidence is particularly convincing. At Tinde laboratory, a strain of T. rhodesiense has been transmitted through sheep by G. morsitans since 1934, and the strain was also passed through various species of antelope for 10 years and monkeys for 6 years (FAIRBAIRN and BUI~TT, 1946 ; WILLETT and FAIRBAIRN, 1955). Human volunteers were infected by single G. morsitans which had fed on infected sheep, monkeys, Thomson's gazelle, impala, dikdik, steinbuck, bushbuck, reedbuck, and bushpig. The strain has retained its infectivity to man throughout the experiment and a volunteer was infected in December, 1957, after the strain had been transmitted through sheep for over 23 years. Various other animals, including eland, duiker and warthog, as well as the species mentioned above, were infected with T. rhodesiense. The infections died out after varying lengths of time but successful fly transmissions were obtained after quite long periods from some animals. For instance, a successflfl fly transmission was obtained 10 months after the initial infection of a reedbuck, and 140 days after the infection of an impala. DuKE (1935, 1937) was able to transmit I". rhodesiense from a hyaena, which had been infected 569 days, and a bushbuck, which had been infected about 600 days, to monkeys and thence to man. Considering these results, it would be very surprising if a T. rhodesiense infection of a wild animal, in natural conditions, never exists. G. pallidipes have been found to carry T. rhodesiense on three occasions (McKIcHAN, 1944 ; E.A.T.R.O. Annual Reports, 1956, and in press) ; it is known that a favoured source of food of G. pallidipes is bushbuck (WEITZ and GLASGOW, 1956), and DUKE (1935, 1937) showed that bushbuck can be infected with T. rhodesiense and carry this trypanosome in a form transmissible to tsetse and infective to man for a long period ; it is therefore highly likely that some bushbuck, in areas where sleeping sickness is carried by G. pallidipes, are infected with T. rhodesiense. Thomson's gazelle, reedbuck and duiker have been infected with strains of T. brucei and the course of the infections was similar to that of T. rhodesiense in the same species (BURTT, pers. commun.). This similarity in infectivity of the two trypanosomes is found in all animals which have been experimentally infected except man ; and as wild animals are a reservoir of T. brucei it is to be expected that they also act as a reservoir of T. rhodesiense. Although the isolation of T. rhodesieme from a wild animal by an investigation such as is reported in this paper would be proof of the existence of naturally infected animals, it would give little information about the importance of the animal reservoir. The importance of the reservoir can be estimated only by epidemiological studies, from which it is extremely difficult to reach incontrovertible conclusions. In order to prove that a man who had contracted sleeping sickness had not acquired the infection from a tsetse which had previously fed on another infected man, that is, to prove that the infection of the tsetse was derived from
M. T. ASRCROFT
281
an animal, one must be certain that no other infected men in the previous 6 m o n t h s - - w h i c h is about the maximum life span of the tsetse - - had passed through the areas in which the man might have contracted the disease. In the conditions of the African bush, with the wandering hunters and fishermen, and considering the sometimes chronic nature of the disease, it is almost impossible to be certain that these conditions are fulfilled. JACKSON (1955), however, described 25 cases of sleeping sickness in his fly-boys living in remote and little visited camps ; he concluded that this was strong evidence that the game was acting as a reservoir because he considered that the chances of the tsetse's transmitting the disease from man to man, in the conditions at these camps, must have been very small indeed. If it is difficult to prove from epidemiological data that wild animals do act as a reservoir of T. rhodesiense, it is even more diflqcult to prove that they do not; such a proof would require certain knowledge that the tsetse vectors in an area in which the disease was present were not feeding on any animals capable of being infected with T. rhodesiense ; this condition is hardly possible as the experiments quoted above have shown that a large number of species can be infected with this trypanosome. In the absence, therefore, of incontrovertible epidemiological proof that the wild animals do, or do not, act as a reservoir of sleeping sickness due to T. rhodesiense, we can only consider the likelihood that they do so, bearing in mind that experiments have shown that many species on which the tsetse feed can be infected. T h e large n u m b e r of cases of the disease which are contracted in the fly belt in the Western Province of Tanganyika, where men are scarce but wild animals are numerous and where the tsetse vector, G. morsitans, is a zoophilic species, suggests that not only is there an animal reservoir but that the part it plays is an important one. In other areas where the disease occurs, the importance of such a reservoir may be less. T h e similarities between T. brucei and T. rhodesiense are so striking, especially their infectivity to all animals except man, that I believe that both are primarily parasites of wild animals with the important and stable difference that T. rhodesiense can also infect man.
The incidence of trypanosomiasis T h e proportion of animals found to be infected with all species of trypanosomes, 10 in 74, (13.5 per cent.) was lower than that found in most previous investigations. KINGHORNand YORKE (1913) found a 22 per cent. incidence in 251 animals in N o r t h e r n Rhodesia ; they used as diagnostic methods the examination of blood films and subinoculations into rats and monkeys. BRUCEet al. (1914) found a 31.7 per cent. incidence in 180 animals in Nyasaland ; diagnosis was by blood films and subinoculations into goats, monkeys and dogs. BUCHANAN (1929) found a 21 per cent. incidence in 150 animals in the Ufipa district of Tanganyika ; diagnosis was by the examination of blood films only. VANDeReLANK (1947) found a 17.5 per cent. incidence in 378 animals from various areas of the Lake Province of Tanganyika : diagnosis was from blood films only. Although the results may represent to some extent true differences between areas, a n u m b e r of factors may influence the incidence found ; these are discussed below. Methods of diagnosis. Neither the examination of blood films nor the subinoculations into rats is a completely satisfactory method. In the present investigation a single trypanosome was seen on some slides, suggesting that more prolonged examination might give a greater n u m b e r of positive findings. Rat subinoculation is of limited value because T. vivax and some strains of T. congolense are unable to infect rats. T h e limitations of both methods
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ATTEMPT TO ISOLATE
Trypanosoma rhodesiense FROM
WILD ANIMALS
are brought out by the fact that, in the present experiment, when an infection was diagnosed from a blood slide, rats were not infected, and vice versa. There is no satisfactory method available for the diagnosis of cryptic trypanosomiasis and it is not impossible that nearly every animal carries trypanosomes, albeit in very small numbers. The species of animal. An analysis of the results of KINGHORN and YORKE (1913), BRUCE et al. (1914), BUCI~ANAN (1929) and VANDERPLANI~ (1947), show that the greatest incidence of trypanosomiasis has been found in waterbuck, bushbuck, reedbuck, kudu, giraffe and eland ; although these six species comprised over 29 per cent. of all the animals examined, they represented 63 per cent. of those found to be infected with trypanosomiasis. Few anim~als of these species were examined in the present investigation and this may be partially responsible for the low incidence found. The season. VANDERPLANK(1947) found a seasonal variation in the number of animals detected with trypanosomiasis, the highest incidence being during the rains. T h e experiments described here took place at the end of the dry season. SUMMARY
1) An attempt was made to isolate strains of polymorphic trypanosomes from wild animals in two areas of endemic T. rhodesiense sleeping sickness in Tanganyika by the subinoculation of blood into rats. One strain of polymorphic trypanosomes was isolated but it did not infect two volunteers. Direct proof of the presence of T. rhodesiense in wild animals is, therefore, still lacking• 2) Blood films from the animals were also examined. Of a total of 74 animals, 10 were found to be infected with T. brucei, T. congolense or T. vivax. The probability is discussed that the true incidence is higher because cryptic infections can be missed. REFERENCES APTED, F. I. C. (1956). Tanganyika Medical Department. Sleeping Sickness Service Annual Report, 1955. BRUCE, D., HARVEY,D., HAMERTON,A. E., DAVEY,J. B. gg LADYBRUCE(1914). Rep. Sleeping Sickness Comm., 15, 16. BUCHANAN,J. C. R. (1929). E. Aft. reed. J., 4, 111. DUNE, H. L. (1935). Parasitology, 25, 171. - (1937). Ibid., 29, 12. East African Trypanosomiasis Research Organisation (1956). Annual Report, 1955-56. • Annual Report, 1956-57 (in press). FAIRBAIRN,H. gg BURTT, E. (1946). Ann. trop. Med. Parasit., 40, 270. JACKSON,C. H. N. (1955). Trans. R. trop. Med. Hyg., 49, 582. KINGHORN,A. 6c YORKE,W. (1913). Ann. trop. Med. Parasit., 7, 184. McKIcI-IAN, J. W. (1944). Trans. R. Soe. trop. Med. Hyg., 38, 49. TAUTE, M. (1913). Abstract in Trop. Dis. Bull., 2, 348. VANDERPLANK,F. L• (1947). Ann. trop. Med. Parasit., 41, 365. WEITZ, B. • GLASGOW,J. P. (1956). Trans. R. Soc. trop. Med. Hyg., 50, 593. WILLETT, K. C. & FAIRBAIRN,It. (1955). Ann. trop. Med. Parasit., 49, 278.