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Studies on Trypanosoma vivax: Transmission of mouse infective T. vivax by tsetse flies
STUDIES ON TRYPANOSOMA VWAX: TRANSMISSION MOUSE INFECTIVE T. VWAX BY TSETSE FLIES
OF
A. L. W. DE GEE,* K. IGEt and P. LEEFLANG$ *Institute for Tropical and Protozoan Diseases, State University of Utrecht, Biltstraat 172, Utrecht, The Netherlands tNigerian Institute for Trypanosomiasis Research, Vom, Benue Plateau State, Nigeria $.Department of Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bell0 University, Zaria, Nigeria (Received 24 September 1975)
Abstract--GEE A. L. W.
DE, IGE K. and LEEFLANG P. 1976. Studies on Trypunosomu vivax: transmission of mouse infective T. vivux by tsetse flies. International Journal for Parasitology 6: 419421. Glossina morsitans and G. tachinoides were successfully infected with 2 ‘isolates of Trypanosoma vivax which had an inherent property for serial maintenance in mice. The infection rate in the flies was relatively high. Cyclical transmission of these isolates from sheep to sheep and from goat to goat was achieved and did not affect the property of the isolates to infect mice. Mice were not apparently suitable for direct fly transmission experiments of 7’. vivax.
INDEX KEY WORDS: Trypanosomu vivax; GIossina morsitans; G. tuchinoides; cattle; sheep; goats; rabbits; mice; infectivity; tsetse transmission; diagnosis; c~oprese~at~on.
INTRODUCTION
in 8% dimethyl sulphoxide (DMSO) and stored at - 196°C for 159 days before use. Similarly, mouse passage 40 from isolate Zaria Y486 was passaged into a Friesian calf. Infected calf blood was deep frozen 25 days after inoculation and stored for 188 days. The G. morsitans population originated from Rhodesia and had been maintained at the Institute for Experimental Entomology in Amsterdam, The Netherlands, since April 1973. Flies of both sexes and of all ages were used. G. tuchinoides was raised from pupae provided by the Institut d’EIevage et de Mddecine V&&inaire des Pays Tropicaux, MaisonsAlfort, France. Male and female flies were used at an age of 1 week. Each isolate in the frozen calf blood was inoculated i.v. into a goat. When positive for T. v&ax, 100 G. morsituns and 70 G. tachinoides were fed on each goat for 3 successive days, before being fed on rabbits for 18 days, and then starved for 1 day. Thereafter, the surviving G. morsitans and G. tachinoides from each donor goat were fed separately on recipient goats for 10 days (Table l), when the then surviving flies were dissected (Table 2). The recipient goats were examined daiIy for trypanosomes by wet blood film examination and mouse-inoculation.
IN A
PREVIOUSpaper, Leeflang, Buys & Blotkamp (1976) described the inherent property of 3 Nigerian isolates of Trypanosoma vivux from naturally infected cattle, for serial maintenance in laboratory mice. In the present paper, the cyclical transmission is reported of 2 of these isolates by Glossinu morsitans and G. ~~c~~~o~~es. MATERIALS
AND METHODS
Zsoiutes of 1”. vivux. The history of the isolates, Zaria Y58 and Zaria Y486, has previously been described (Leeflang et al., 1976). Experimental procedures. Experiment 1 (The Netherlands). Laboratory bred white Swiss mice and rabbits, and cross-bred l-year-old dwarf goats were used. Mouse passage 21 from isolate Zaria Y58 was sent from Nigeria and inocuiated i.v. into a Friesian calf. Seven days after inoculation, the T. vivux positive blood was deep frozen
Experiment 1 1 2 1 1
TABLE~-THE TRANSMISSION OF MOUSE-INFECTIVE Trypunosoma vivax BY Glossina SPECIES Recipient animals First day wet blood film positive after Prepatent period Prepatent period first exposure of flies by wet blood by mouse T. vivax isolate Species of Glossina to trypanosomes film examination inoculation Y58 Y58 Y58 Y486 Y486
G. G. G. G. G.
morsituns tacfiinoides tachi~oides morsitans tachinoides
*Not done. 419
31 30 33 31 31
9 8 17 9 9
6 6 N.D.* 6 6
420
A. L. W.
DE GEE,
K.
IGE and
P.
TABLE ~-RESULTS OF TSETSEFLY DISSECTIONFOR THE PRESENCE OF
Experiment
T. vivux isolate Y58 Y58 Y486 Y486 Y486
Species of Glossina G. G. G. G. G.
morsitans tachinoides morsitans tachinoides tachinoides
Experiment 2 (Nigeria). West African Dwarf sheep and white rabbits, laboratory bred and free of trypanosomiasis were used. Infected blood from mouse passage 129 of isolate Zaria Y58 and from passage 48 of isolate Zaria Y486 were inoculated i.v., separately, into 2 sheep. When the sheep became positive for T. vivax, 25 teneral flies of G. tachinoides were allowed to feed on each sheep for 5 successive days. The flies had been obtained from pupae, collected at Yankari Game Reserve, Nigeria. After feeding on the infected sheep, the flies were starved for 1 day, fed on rabbits for 9 days, starved for 1 day, and then fed daily on uninfected sheep. The recipient sheep for isolate Zaria Y486 died before T. vivax could be
demonstrated. The surviving flies were then dissected and examined for the presence of T. vivax (Table 2). RESULTS Both isolates were transmitted by G. morsitans and G. tuchinoides (Table 1). The period between first exposure of the flies to trypanosomes and the first day of detectable trypanosomes in wet blood films of the recipient animals was about the same in both experiments. Difference in the transmission procedures in experiments 1 and 2 is reflected in the different length of the prepatent period in the recipient animals used in each experiment. The advantage of mouse-inoculation as a method to diagnose mouse-infective T. vivax was clearly demonstrated when compared with the results of wet blood film examination (Table 1). In the recipient goats, the isolates remained infective to mice for a period of 35 days, when inoculation was discontinued. Mouse-inoculation was successful on days when trypanosomes could not be detected in wet blood films. Table 2 shows the results of tsetse fly dissection; trypanosomes were observed only in the probosces of the flies at a relatively high infection rate which is a characteristic of T. vivax. Triturated infected probosces from both G. morsitans and G. tachinoides, when inoculated into mice, did not result in apparent infection. DISCUSSION Desowitz & Watson (1953) transmitted their ratadapted isolate of T. vivax from rat to sheep using G. palpalis, with the consequence that the T. vivax from the sheep lost its ability to be maintained in
I.J.P. VOL. 6. 1976
LEEFLANG
Days after first fly exposure to trypanosomes 32 32 32 32 29
Trypanosoma vivax Number dissected 18 5 27 11 9
Number positive 3 3 16 7 8
rats. At a later stage of serial maintenance in rats, it was only possible to obtain an occasional infected fly. Hull, Swain, McCabe, Jones & Clarkson (1971) failed to infect G. morsitans with their rat-adapted isolate for T. vivax. Hull (1972) was successful in the transmission of the same isolate, adapted to splenectomized rabbits, by G. morsitans from rabbit to rabbit, but only when sheep serum supplement was inoculated. The present isolates of T. vivax were infective to 2 species of tsetse flies and were also transmissible cyclically from sheep to sheep and from goat to goat. Using G. morsitans, Dr. B. Elce of Brunel University has not been able to transmit isolate Zaria Y486 mouse-fly-mouse, although the probosces were found to be infected. Like our experience, the direct inoculation of metatrypanosomes failed to infect mice. The mouse, thus, appears to be unsuitable for direct transmission experiments. Infectivity for mice remained after fly transmission (Table 1) which confirms that the isolates had an inherent property to infect mice. Although thismouseinfective T. vivax may not provide a mouse-flymouse model, its unchanged characteristics during both, serial maintenance in mice and cyclical transmission by a mouse-ruminant-fly-ruminant-mouse cycle, offer an increased opportunity to institute more fundamental research on this trypanosome species. Acknowledgements-The authors are most grateful to Dr. B. Elce of Brunel University, England, for his permission to include the results of his transmission experiments in this paper; to the Institute for Experimental Entomology, Amsterdam, the Netherlands, in particular to Ir. L. P. S. van der Geest for advice, assistance and the supply of G. morsitans; to the Institut d’Elevage et de MCdecine VBt&inaire des Pays Tropicaux, MaisonsAlfort, France, for the supply of G. tuchinoides pupae; to the staff of the various institutions that participated in this work, and to Dr. D. G. Godfrey for reading the manuscript. K. Ige thanks the Director of the Nigerian Institute for Trypanosomiasis Research for his permission to publish. REFERENCES DESOWITZ R. S. & WATSON H. J. C. 1953. Studies on
Trypanosoma vivax. IV. The maintenance of a strain in white rats without sheep-serum supplement. Annals of Tropical Medicine and Parasitology 47: 62-67.
I.J.P. VOL.6. 1976
Transmission
of mouse infective T. vivax
HULL R. M. 1972. Cyclical transmission of a WestAfrican strain of Trypunosoma vivax in rabbits. Transactions of the Royal Society of Tropical Medicine and Hygiene 66: 334-335. HULL R. M., SWAIN F., MCCABE W., JONES T. W. & CLARKSONM. J. 1971. Adaptation of Trypanosoma
421
vivax to laboratory animals. Transactions of the Royal Society of TropicaI Medicine and Hygiene 65: 14-15. LEEFLANGP., BUYS, JANNY & BLOTKAMP,COBY. 1976. Studies on Trypanosoma vivux: infectivity and serial maintenance of natural bovine isolates in mice. Znternational Journal for Parasitology 6: 413-417.
OF
A. L. W. DE GEE,* K. IGEt and P. LEEFLANG$ *Institute for Tropical and Protozoan Diseases, State University of Utrecht, Biltstraat 172, Utrecht, The Netherlands tNigerian Institute for Trypanosomiasis Research, Vom, Benue Plateau State, Nigeria $.Department of Parasitology and Entomology, Faculty of Veterinary Medicine, Ahmadu Bell0 University, Zaria, Nigeria (Received 24 September 1975)
Abstract--GEE A. L. W.
DE, IGE K. and LEEFLANG P. 1976. Studies on Trypunosomu vivax: transmission of mouse infective T. vivux by tsetse flies. International Journal for Parasitology 6: 419421. Glossina morsitans and G. tachinoides were successfully infected with 2 ‘isolates of Trypanosoma vivax which had an inherent property for serial maintenance in mice. The infection rate in the flies was relatively high. Cyclical transmission of these isolates from sheep to sheep and from goat to goat was achieved and did not affect the property of the isolates to infect mice. Mice were not apparently suitable for direct fly transmission experiments of 7’. vivax.
INDEX KEY WORDS: Trypanosomu vivax; GIossina morsitans; G. tuchinoides; cattle; sheep; goats; rabbits; mice; infectivity; tsetse transmission; diagnosis; c~oprese~at~on.
INTRODUCTION
in 8% dimethyl sulphoxide (DMSO) and stored at - 196°C for 159 days before use. Similarly, mouse passage 40 from isolate Zaria Y486 was passaged into a Friesian calf. Infected calf blood was deep frozen 25 days after inoculation and stored for 188 days. The G. morsitans population originated from Rhodesia and had been maintained at the Institute for Experimental Entomology in Amsterdam, The Netherlands, since April 1973. Flies of both sexes and of all ages were used. G. tuchinoides was raised from pupae provided by the Institut d’EIevage et de Mddecine V&&inaire des Pays Tropicaux, MaisonsAlfort, France. Male and female flies were used at an age of 1 week. Each isolate in the frozen calf blood was inoculated i.v. into a goat. When positive for T. v&ax, 100 G. morsituns and 70 G. tachinoides were fed on each goat for 3 successive days, before being fed on rabbits for 18 days, and then starved for 1 day. Thereafter, the surviving G. morsitans and G. tachinoides from each donor goat were fed separately on recipient goats for 10 days (Table l), when the then surviving flies were dissected (Table 2). The recipient goats were examined daiIy for trypanosomes by wet blood film examination and mouse-inoculation.
IN A
PREVIOUSpaper, Leeflang, Buys & Blotkamp (1976) described the inherent property of 3 Nigerian isolates of Trypanosoma vivux from naturally infected cattle, for serial maintenance in laboratory mice. In the present paper, the cyclical transmission is reported of 2 of these isolates by Glossinu morsitans and G. ~~c~~~o~~es. MATERIALS
AND METHODS
Zsoiutes of 1”. vivux. The history of the isolates, Zaria Y58 and Zaria Y486, has previously been described (Leeflang et al., 1976). Experimental procedures. Experiment 1 (The Netherlands). Laboratory bred white Swiss mice and rabbits, and cross-bred l-year-old dwarf goats were used. Mouse passage 21 from isolate Zaria Y58 was sent from Nigeria and inocuiated i.v. into a Friesian calf. Seven days after inoculation, the T. vivux positive blood was deep frozen
Experiment 1 1 2 1 1
TABLE~-THE TRANSMISSION OF MOUSE-INFECTIVE Trypunosoma vivax BY Glossina SPECIES Recipient animals First day wet blood film positive after Prepatent period Prepatent period first exposure of flies by wet blood by mouse T. vivax isolate Species of Glossina to trypanosomes film examination inoculation Y58 Y58 Y58 Y486 Y486
G. G. G. G. G.
morsituns tacfiinoides tachi~oides morsitans tachinoides
*Not done. 419
31 30 33 31 31
9 8 17 9 9
6 6 N.D.* 6 6
420
A. L. W.
DE GEE,
K.
IGE and
P.
TABLE ~-RESULTS OF TSETSEFLY DISSECTIONFOR THE PRESENCE OF
Experiment
T. vivux isolate Y58 Y58 Y486 Y486 Y486
Species of Glossina G. G. G. G. G.
morsitans tachinoides morsitans tachinoides tachinoides
Experiment 2 (Nigeria). West African Dwarf sheep and white rabbits, laboratory bred and free of trypanosomiasis were used. Infected blood from mouse passage 129 of isolate Zaria Y58 and from passage 48 of isolate Zaria Y486 were inoculated i.v., separately, into 2 sheep. When the sheep became positive for T. vivax, 25 teneral flies of G. tachinoides were allowed to feed on each sheep for 5 successive days. The flies had been obtained from pupae, collected at Yankari Game Reserve, Nigeria. After feeding on the infected sheep, the flies were starved for 1 day, fed on rabbits for 9 days, starved for 1 day, and then fed daily on uninfected sheep. The recipient sheep for isolate Zaria Y486 died before T. vivax could be
demonstrated. The surviving flies were then dissected and examined for the presence of T. vivax (Table 2). RESULTS Both isolates were transmitted by G. morsitans and G. tuchinoides (Table 1). The period between first exposure of the flies to trypanosomes and the first day of detectable trypanosomes in wet blood films of the recipient animals was about the same in both experiments. Difference in the transmission procedures in experiments 1 and 2 is reflected in the different length of the prepatent period in the recipient animals used in each experiment. The advantage of mouse-inoculation as a method to diagnose mouse-infective T. vivax was clearly demonstrated when compared with the results of wet blood film examination (Table 1). In the recipient goats, the isolates remained infective to mice for a period of 35 days, when inoculation was discontinued. Mouse-inoculation was successful on days when trypanosomes could not be detected in wet blood films. Table 2 shows the results of tsetse fly dissection; trypanosomes were observed only in the probosces of the flies at a relatively high infection rate which is a characteristic of T. vivax. Triturated infected probosces from both G. morsitans and G. tachinoides, when inoculated into mice, did not result in apparent infection. DISCUSSION Desowitz & Watson (1953) transmitted their ratadapted isolate of T. vivax from rat to sheep using G. palpalis, with the consequence that the T. vivax from the sheep lost its ability to be maintained in
I.J.P. VOL. 6. 1976
LEEFLANG
Days after first fly exposure to trypanosomes 32 32 32 32 29
Trypanosoma vivax Number dissected 18 5 27 11 9
Number positive 3 3 16 7 8
rats. At a later stage of serial maintenance in rats, it was only possible to obtain an occasional infected fly. Hull, Swain, McCabe, Jones & Clarkson (1971) failed to infect G. morsitans with their rat-adapted isolate for T. vivax. Hull (1972) was successful in the transmission of the same isolate, adapted to splenectomized rabbits, by G. morsitans from rabbit to rabbit, but only when sheep serum supplement was inoculated. The present isolates of T. vivax were infective to 2 species of tsetse flies and were also transmissible cyclically from sheep to sheep and from goat to goat. Using G. morsitans, Dr. B. Elce of Brunel University has not been able to transmit isolate Zaria Y486 mouse-fly-mouse, although the probosces were found to be infected. Like our experience, the direct inoculation of metatrypanosomes failed to infect mice. The mouse, thus, appears to be unsuitable for direct transmission experiments. Infectivity for mice remained after fly transmission (Table 1) which confirms that the isolates had an inherent property to infect mice. Although thismouseinfective T. vivax may not provide a mouse-flymouse model, its unchanged characteristics during both, serial maintenance in mice and cyclical transmission by a mouse-ruminant-fly-ruminant-mouse cycle, offer an increased opportunity to institute more fundamental research on this trypanosome species. Acknowledgements-The authors are most grateful to Dr. B. Elce of Brunel University, England, for his permission to include the results of his transmission experiments in this paper; to the Institute for Experimental Entomology, Amsterdam, the Netherlands, in particular to Ir. L. P. S. van der Geest for advice, assistance and the supply of G. morsitans; to the Institut d’Elevage et de MCdecine VBt&inaire des Pays Tropicaux, MaisonsAlfort, France, for the supply of G. tuchinoides pupae; to the staff of the various institutions that participated in this work, and to Dr. D. G. Godfrey for reading the manuscript. K. Ige thanks the Director of the Nigerian Institute for Trypanosomiasis Research for his permission to publish. REFERENCES DESOWITZ R. S. & WATSON H. J. C. 1953. Studies on
Trypanosoma vivax. IV. The maintenance of a strain in white rats without sheep-serum supplement. Annals of Tropical Medicine and Parasitology 47: 62-67.
I.J.P. VOL.6. 1976
Transmission
of mouse infective T. vivax
HULL R. M. 1972. Cyclical transmission of a WestAfrican strain of Trypunosoma vivax in rabbits. Transactions of the Royal Society of Tropical Medicine and Hygiene 66: 334-335. HULL R. M., SWAIN F., MCCABE W., JONES T. W. & CLARKSONM. J. 1971. Adaptation of Trypanosoma
421
vivax to laboratory animals. Transactions of the Royal Society of TropicaI Medicine and Hygiene 65: 14-15. LEEFLANGP., BUYS, JANNY & BLOTKAMP,COBY. 1976. Studies on Trypanosoma vivux: infectivity and serial maintenance of natural bovine isolates in mice. Znternational Journal for Parasitology 6: 413-417.