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Malic enzyme type VII isoenzyme as an indicator of suramin resistance in Trypanosoma evansi
EXPERIMENTAL PARASITOLOGY 69, 317-323 (1989)
Malic Enzyme Type VII lsoenzyme as an Indicator of Suramin Resistance in TrVpanosoma evansi R. BOID,* T. W. JONES,*AND R. C. PAYNE' Centre for Tropical
Veterinary
Medicine,
Easter Bush, Roslin, Midlothian,
EH25 9RG, Scotland
BOID, R., JONES, T. W., AND PAYNE, R. C. 1989. Malic enzyme type VII isoenzyme as an indicator of suramin resistance in Trypanosoma evansi. Experimental Parasitology 69: 317-323. This study analysed the surarnin sensitivity of 29 stocks of Trypanosoma evansi isolated from Egypt, Sudan, and Indonesia and compared the results with the isoenzyme banding patterns of 20 soluble enzymes in these stocks of T. evansi. The results showed that the type VII banding pattern of malic enzyme was found only in T. evansi stocks which were highly resistant to suramin. 8 1989Academic press, IIIC. INDEX DESCRIPTORSAND ABBREVIATIONS: Trypanozoon; Trypanosoma evansi; Suramin; Drug resistance; Isoenzyme; Starch gel electrophoresis; Phosphate-buffered saline glucose (PBSG); Malic enzyme (ME); Nicotinamide adenine dinucleotide phosphate (NADP); Thiazolyl blue (MTT). High powered field (HPF).
Indonesia (Kraneveld and Mansjoer 1941). These few reports probably understate the Trypanosomiasis caused by Trypanoreal extent of suramin resistance in T. soma evansi (surra) affects domestic live- evunsi since cases of suramin resistance are stock in countries from South America only infrequently reported in the literature through North Africa to the Middle and Far (Mahmoud and Gray 1980). East of Asia (Mahmoud and Gray 1980). To make more effective use of the availThe trypanosome is transmitted by several able trypanocides and to limit the spread of species of blood-sucking dipterous flies resistance to suramin, there is an urgent which makes controlling the disease by in- need for a method to detect, at an early secticide spraying impracticable. The only stage, the emergence of T. evunsi stocks effective method of dealing with sun-a is the resistant to suramin, particularly in counuse of chemotherapy. This depends on only tries where suramin is used intensively. four drugs: suramin, quinapyramine sul- The existing method of screening T. evunsi fate, isometamidium chloride, and dimina- stocks for drug resistance depends on obzene aceturate (Boid et al. 1985). In prac- serving the response of T. evunsi-infected tice, suramin is the drug used for treatment laboratory rodents to different amounts of of T. evunsi-infected animals. Suramin has the drug. Such a method is labour intenbeen in constant use as a trypanocide since sive, slow, and expensive. In order to im1925 (Williamson 1976) and reports of prove the existing methods, we looked for a suramin-resistant strains of T. evunsi are in- correlation between the isoenzyme banding creasing. Such strains have been reported patterns of 20 enzymes and suramin sensifrom Sudan (Leach 1961; Luckins et al. tivity in 29 stocks of T. evunsi isolated in 1979; Abebe et al. 1983), India (Gill 1971), North Africa and Indonesia. In this paper Russia (Petrovskii and Khamiev 1974), and we present evidence for a relationship between the suramin resistance of T. evansi stocks and the presence of a type VII isoenzyme (Gibson et al. 1980; Boid 1988) of r Present address: Balitvet, JaIan R. E. Martadinata malic enzyme. 32, P. 0. Box 52, Bogor, Java, Indonesia. INTRODUCTION
317 0014-4894/89$3.00 Copyright All riahts
0 1989 by Academic Press, Inc. of reoroduction in anv form reserved.
318
BOID,JONES, AND PAYNE MATERIALSAND
METHODS
Suramin. Suramin B. P. (Antrypol, ICI Ltd., Wilmslow, Cheshire) was used in this study as a sterile white powder. Solutions for injection were freshly prepared by dissolving the required amount of the drug in sterile distilled water at room temperature. Laboratory animals. Random-bred female albino CFl mice weighing 25 to 30 g were obtained from the
Centre for Laboratory Animals, University of Edinburgh. Ttypanosomes. AU trypanosome stocks used in this study were considered to represent examples of T. brucei evansi according to Hoare (1972). Trypanosome stocks were originally isolated from Egypt (1), Sudan (15), and Indonesia (13). Isolation details of the stocks are shown in Table I and Fig. 1. All trypanosome populations were derived from a single cloned
TABLE I T. evansi Stocks: Origin, Degree of Resistance to Suramin, and Malic Enzyme Type
Isolation details Stock no.
Host
Town
Country
Year
Group 1 1. T. evansi stock with type 1 suramin resistance and type II malic enzyme isoenzymes 1 Camel Soba Sudan Camel Kassala Sudan 2 Camel Kassala Sudan 3 Kassala Sudan 4 Camel Bovine ? Bali 5 Minahasa N. Sulawesi Bovine 6 Bovine Bolamongondo N. Sulawesi 7 Mlangi 8 Bovine E. Java Bovine Bogor W. Java 9 Telaga Mumi W. Java 10 Bovine Buffalo Gowa S. Sulawesi 11 Mlangi Buffalo E. Java 12 Buffalo Pekalongan C. Java 13 Ciawi W. Java 14 Buffalo Buffalo Bogor W. Java 15 Buffalo Bekasi W. Java 16 W. Java Buffalo Bogor 17
1977 Jan. 1977 1977 1977 1978 1984 1982 1983 1983 1983 1984 1983 1982 1984 1982 1984 1983
Group 2 2. T. evnnsi stock with type 2 suramin resistance and type II malic enzyme isoenzymes Shambat Sudan Camel 18 clone Shambat Sudan Camel 18 clone Sudan Camel Urn Hagar 19 clone Camel Urn Hagar Sudan 19 clone Camel Urn Gegeim Sudan 20 clone Urn Gegeim Sudan 20 clone Camel Sudan Camel Urn Gegeim 21 clone Urn Gegeim Sudan 21 clone Camel
Dec. Dec. Mar. Mar. Mar. Mar. Mar. Mar.
1976 1976 1977 1977 1977 1977 1977 1977
Oct. Oct. Oct. Apr. Apr. Jan. Jan.
1978 1978 1978 I978 1978 1977 1977 ?
Group 3 3. T. evansi stock with type 3 suramin resistance and type VII malic enzyme isoenzymes Soba Sudan Camel 22 Camel Soba Sudan 23 Camel Soba Sudan 24 Sudan 25 Camel Hadaliya 26 Camel Hadaliya Sudan 27 Camel Aroma Sudan 28 Camel Aroma Sudan Camel Cairo 29 Egypt
T.
evansi:
SURAMIN
30’
25’ I
SENSITIVITY
35’
EGYPT
A. FL LIBYA r
EHAD
SUDAN
___------Tsetse
Area
FIG. 1. Locations in Kassala Province, Sudan from which stocks of T. evansi were isolated.
population of each stock prepared by the method of Gardiner et al. (1980). A second cloned population was prepared from each of the stocks numbered 18, 19,20, and 21. Testing for drug sensitivity. Before use, stabilate material of each clone was expanded in mice which were bled at peak parasitaemia (4-6 days after infection) and the trypanosomes were separated from host erythrocytes by ion-exchange chromatography (Lanham and Godfrey 1970).
Column-separated trypanosomes of each stock were washed twice with PBSG, counted using a haemocytometer, and diluted with PBSG at pH 8.0 to give a final concentration of 5 X lo6 trypanosomeslml. Groups of five mice were used for each trypanosome stock at each suramin dose level. AU mice were inoculated intraperitoneally with 1 x lo6 trypanosomes. All living mice were examined for parasites daily, using wet films prepared from tail blood, for a period of 31 days after infection.
320
BOID, JONES, AND PAYNE
Twenty-four hours after infection, four mice from each group were injected intraperitoneally with 0.2 ml suramin at dose rates of 4,8, 16,32, or 64 mg/kg. The fifth mouse from each group acted as a control for that group and did not receive suramin.
stocks 18 to 21 inclusive were considered to be resistant to 4 mg/kg of suramin; parasitaemias in the mice receiving this dose were similar to those in control mice 4-8 days after treatment. Treatment of mice infected Extraction of malic enzyme (ME) (EC 1 .I .1.40) from trypanosomes. Trypanosomes representing each stock with any one of these four T. evansi stocks employed in the present study were separated from with suramin at dose rates of either 8 or 16 infected mouse blood by ion-exchange chromatography (Lanham and Godfrey 1970). The soluble en- mg/kg also did not eliminate the infection; zymes, including ME, were extracted from these rep- some of the mice developed sporadic pararesentative trypanosome populations and stored as sitaemias of between ~1 and lOO/HPF dur2&l beads in liquid nitrogen as described by Gibson ing the 31-day observation period. Mice inet al. (1978). fected with any one of these four stocks and Electrophoretic separation of malic enzyme. The electrophoretic mobility of ME was examined using then treated with suramin at a dose of 32 horizontal thin-layer starch gel electrophoresis using mg/kg became aparasitaemic 24-48 hr after the method of Wraxall and Culliford (1968) in 0.7- treatment and remained so for the rest of mm-thick gels using a starch concentration of 10.6% the 31-day observation period. New cloned w/v. Electrophoresis was carried out at 300 V and at a populations were prepared from each of temperature of 8°C for 3 hr using a 0.2 M phosphate these four stocks and the experiment was buffer, pH 7.4, system (Boid 1988). Malic enzyme bands were visualized with a stain repeated with one example of each new solution containing 10 ml 0.5 M Tris/HCl buffer, pH clone; similar results were obtained. 7.4; 2 ml 0.1 M L-malate, pH 7.0; 2 ml MTT (4 mg/mI); Group 3: Mice infected with T. evansi 100 pl meldola blue (6 mg/ml); 1 ml NADP (10 mg/ml); stocks 22 to 29 inclusive differed markedly mixed with 10 ml warm agar solution (70 mg/ml). Gels in their reaction to suramin when compared were incubated in the dark at 37°C for 30 min. The nomenclature used for the isoenzyme banding patterns with the other stocks tested in groups 1 and of malic enzyme is that used by Gibson et al. (1980). 2. Mice infected with any one of the eight stocks and then treated with suramin at all RESULTS doses of up to 64 mg/kg all reached parasitaemias of lOO/HPF within 3 to 5 days after Suramin sensitivity of T. evansi stocks treatment. Control mice. Trypanosomes appeared in all mice 24 hr after infection and parasi- Isoenzymes taemias increased progressively until 100 T. evansi stocks 1 to 21 inclusive were trypanosomes/HPF were present 4 to 8 found to have type II isoenzyme banding days after infection. Control mice generally patterns for malic enzyme. The eight other died with a fulminating parasitaemia 8 to 18 T. evansi stocks, 22 to 29 inclusive, had a days after infection. type VII isoenzyme banding pattern for Suramin-treated mice. These results are malic enzyme (Table I). summarised in Table I. Trypanosome The two enzyme banding patterns are ilstocks were categorized into groups 1, 2, lustrated diagrammatically in Fig. 2. Bands and 3 according to their degree of resis- b and c are present in the type II pattern but are absent from the type VII pattern. The tance to suramin. Group 1: Mice infected with T. evansi absence of any significant enzymic activity stocks 1 to 17 inclusive were all trypano- at positions b and c was confirmed by loadsome negative 24 hr after injection of ing gels with fivefold concentrated samples. suramin at a dose of 4 mg/kg. Thereafter no DISCUSSION trypanosomes were seen in the blood during the observation period of 31 days. The 29 trypanosome stocks examined in Group 2: Mice infected with T. evansi this work showed a range of sensitivities to
T. evansi: SURAMIN SENSITIVITY Type II
Type VII +
Cba-
-
-a
-
origin
FIG. 2. Diagram of the type II and type VII enzyme banding patterns of malic enzyme from stocks of T. evansi. n , strong enzyme activity. Each enzyme band is designated by a lowercase letter.
the trypanocide suramin. Trypanosome stocks belonging to group 1 were sensitive to the normal dose of suramin (4 mg/kg) required to clear suramin-sensitive stocks of T. evansi from infected mouse blood (Abebe et al. 1983). Stocks ascribed to group 2 were less sensitive than group 1 stocks and required suramin at dose rates of between 16 and 32 mg/kg to clear them from mouse blood. Group 3 trypanosome stocks were highly resistant to suramin at dose rates of up to 64 mg/kg. Correlation of the suramin resistance of the previous groups with the isoenzyme banding patterns of malic enzyme showed that all T. evansi stocks which had some degree of suramin sensitivity (groups 1 and 2) exhibited a type II malic enzyme banding pattern (see Table I). Group 3 stocks, which were highly resistant to suramin, had a type VII enzyme banding pattern. Three isoenzyme banding patterns of malic enzyme, types II, VII, and XXIV, have been reported to occur in T. evansi (Gibson et al. 1980;Boid 1988).The type II pattern represents the most frequently encountered banding pattern for this enzyme in T. evunsi and has been reported in stocks isolated from Colombia, Nigeria, Kenya, Sudan, and Indonesia (Gibson et al. 1980, 1983; Boid 1988). Only one example of the
321
type XXIV pattern has been reported (Gibson et al. 1983).The type VII malic enzyme banding pattern has only been reported from eight stocks of T. evunsi (Boid 1988). Two of the eight stocks were isolated at Aroma in 1977;two stocks were isolated at Hadaliya, some 50 km north of Aroma, in 1978; three stocks were isolated at Soba, some 300 km to the west, in 1978; and one stock was obtained from Cairo, Egypt (Fig. 1). The nomenclature used for the isoenzyme banding patterns of malic enzyme is consistent with that used by Gibson et al. (1980) for members of the subgenus Trypunozoon.
The results of this study suggest that stocks of T. evunsi which are found to have a type VII malic enzyme banding pattern may be highly suramin resistant (>64 mg/ kg). Previous workers have examined stocks of T. brucei and failed to establish a correlation between particular isoenzyme banding patterns (including malic enzyme) and resistance to the trypanocidal drugs trypamidium, melarsoprol, pentamidine, and berenil (Dukes 1984). Isoenzymically defined West African stocks were shown to be more sensitive to pentamidine and berenil than East African stocks. No experimental evidence has been published to explain the trypanocidal action of suramin on T. evunsi. The results of this study suggest that the trypanocidal action of suramin against T. evunsi may be due, at least in part, to the inhibition of one or more isoenzymes of malic enzyme. A preliminary investigation has been carried out on unpurified enzyme preparations from one suramin-sensitive T. evunsi (stock 2, Table I) and one suramin-resistant T. evunsi (stock 28, Table I). Under the conditions of the assay, 20 @4 suramin inhibited the activity of malic enzyme in the suraminsensitive stock by approximately twice as much as it inhibited the enzyme in the suramin-resistant stock (R. Boid, unpublished). The action of suramin on the isoen-
322
BOID,JONES, AND PAYNE
zyme types of suramin is being further investigated. Of the T. evansi stocks examined in this work, stocks that were highly resistant to suramin were only found in Northeast Africa and not in Indonesia. This probably reflects the constant exposure of trypanosome-infected animals to chemotherapeutic treatment in these countries, which has undoubtedly led to the spread of drugresistant strains. Despite this, there is no detailed information on drug resistance in T. evansi (Schillinger 1984)and there are no simple tests to determine the drug sensitivity of trypanosome stocks isolated in the field. The identification of malic enzyme as a marker for suramin resistance in stocks of T. evansi should eventually allow the development of an enzyme-linked immunosorbent assay to detect suramin-resistant stocks of T. evansi. The availability of such a test would let the veterinarian select alternative chemotherapeutic regimes to control infection and limit the spread of suramin resistance. ACKNOWLEDGMENTS The authors thank Dr. P. Stevenson, Mr. A. Day,
BPPH, for isolating the trypanosome stocks in Indonesia, and Dr. A. J. Wilson and Dr. H. R. Pumomo, Director, BPPH, for providing facilities in Indonesia. The assistance of the Dean of the Faculty of Veterinary Science, University of Khartoum, and the cooperation of the staff of the Sudan Ministry of Agriculture and Natural Resources is gratefully acknowledged. Professor Dr. F. Hiirchner, Institut fur parasitologie und tropenveterinarmedizin, Frae Universitat Berlin, kindly provided the Egyptian stock of T. evansi. Financial assistance was provided by the Overseas Development Administration of the United Kingdom.
REFERENCES ABEBE, G., JONES, T. W., AND BOID, R. 1983. Suramin sensitivity of stocks of Trypanosoma evansi isolated in the Sudan. Tropical Animal Health and Producrion 15, 151-152. BOID, R. 1988. Isoenzyme characterisation of 15 stocks of Trypanosoma evansi isolated from camels in the Sudan. Tropical Medicine and Parasiiology 39,45-50.
BOID, R., JONES,T. W., AND LUCKINS, A. G. 1985. Protozoal diseases of camels (a review). British Veterinary Journal 141, 87-105. DUKES, P. 1984. Arsenic and old taxa: Subspeciation and drug sensitivity in Trypanosoma brucei. Transactions of rhe Royal Society of Tropical and Hygiene 18, 711-725.
Medicine
FAIRLAMB, A. H., AND BOWMAN, I. B. R. 1977. Inhibitor studies on particulate sn-glycerol phosphate oxidase from Trypanosoma brucei. Znrernarional Journal
of Biochemistry
8, 668-675.
GARDINER,P. R., JONES,T. W., AND CUNNINGHAM, I. 1980. Antigenic analysis by immunofluorescence of in vitro-produced metacyclics of Trypanosoma brucei and their infections in mice. Journal of Prorozoology 27, 316320. GIBSON, W. C., deC. MARSHALL, T. F., AND GODFREY, D. G. 1980. Numerical analysis of enzyme polymorphism: A new approach to the epidemiology and taxonomy of the subgenus Trypanozoon. In “Advances in Parasitology” (W. H. Lumsden, R. Muller, and J. R. Baker, Eds.), pp. 175-246. Academic Press, New York/London. GIBSON,W. C., PARR,C. W., SWINDLEHURST,C. A., AND WELSH, S. G. 1978.A comparison of the isoenzymes, proteins, polypeptides and free amino acids from ten isolates of Trypanosoma evansi. Comparative Biochemistry and Physiology B60, 137-142. GIBSON, W. C., WILSON, A. J., AND MOLOO, S. K. 1983. Characterisation of Trypanosoma (Trypanozoon) evansi from camels in Kenya using isoenzyme electrophoresis. Research in Veterinary Science 34, 114-118. GILL, B. S. 1971. Drug resistance in Trypanosoma evansi. Tropical
Animal
Health
and Production
3,
195-198. HOARE, C. A. 1972. “The Trypanosomes of Mammals: A Zoological Monograph.” Blackwell, Oxford. KRANEVELD, F. C., AND MANSJOER,M. 1941. Proeven over vervangproduction voor Naganol Bayer en Arsocoll. I. Naganol SPECIA. Nederlandsch Zndische Bladen voor Diergeneeskunde
53, 465-475.
LANHAM, S. M., AND GODFREY,D, G, 1970.Isolation of salivarian trypanosomes from man and other mammals using DEAE cellulose. Experimental Parasitology 28, 521-534. LEACH, T. H. 1961. Observations in the treatment of Trypanosoma evansi infection in camels. Journal of Comparative Pathology and Therapeutics 71, 109117. LUCKINS, A. G., BOID, R., RAE, P. F., MAHMOUD, M. M., ELMALIK, K. H., AND GRAY, A. R. 1979. Serodiagnosis of infection with T. evansi in camels in the Sudan. Tropical Animal Health and Production 11, 1-17. MAHMOUD, M. M., AND GRAY, A. R. 1980. Trypano-
T. evansi: SURAMINSENSITIVITY somiasis due to Trypanosoma evansi (Steel, 1885) Balbiani, 1888: A review of recent research. Tropical Animal Health and Production 12, 3547. MESCHNICK, S. R. 1984. The chemotherapy of African trypanosomiasis. In “Parasitic Diseases,” Vol. 2, “The Chemotherapy” (John M. Mansfield, Ed.), pp. 171-172, Decker, New YorWBasel. F’ETROVSKII, V. V., AND KHAMIEV, S. K. M. 1974. Resistance to naganin (suramin) of strains of trypanosomes isolated from camels. Veterinariya 4, 8384. SCHILLINGER, D. 1984. The problem of drug resis-
323
tance. In Proceedings of the KVA Meeting of “Field Aspects of Trypanosomiasis in Kenya,” Mombasa, August 9-10, 1984. WILLIAMSON, J. 1976. Chemotherapy of African trypanosomiasis. Tropical Diseases Bulletin 73,(7), 53l-542. WRAXALL, B. G. D., AND CULLIFORD, B. J. 1968. A
thin-layer starch-gel method for enzyme typing of blood stains. Journal of the Forensic Science Society 8, 81. Received 5 October 1988; accepted with revision 15 May 1989
Malic Enzyme Type VII lsoenzyme as an Indicator of Suramin Resistance in TrVpanosoma evansi R. BOID,* T. W. JONES,*AND R. C. PAYNE' Centre for Tropical
Veterinary
Medicine,
Easter Bush, Roslin, Midlothian,
EH25 9RG, Scotland
BOID, R., JONES, T. W., AND PAYNE, R. C. 1989. Malic enzyme type VII isoenzyme as an indicator of suramin resistance in Trypanosoma evansi. Experimental Parasitology 69: 317-323. This study analysed the surarnin sensitivity of 29 stocks of Trypanosoma evansi isolated from Egypt, Sudan, and Indonesia and compared the results with the isoenzyme banding patterns of 20 soluble enzymes in these stocks of T. evansi. The results showed that the type VII banding pattern of malic enzyme was found only in T. evansi stocks which were highly resistant to suramin. 8 1989Academic press, IIIC. INDEX DESCRIPTORSAND ABBREVIATIONS: Trypanozoon; Trypanosoma evansi; Suramin; Drug resistance; Isoenzyme; Starch gel electrophoresis; Phosphate-buffered saline glucose (PBSG); Malic enzyme (ME); Nicotinamide adenine dinucleotide phosphate (NADP); Thiazolyl blue (MTT). High powered field (HPF).
Indonesia (Kraneveld and Mansjoer 1941). These few reports probably understate the Trypanosomiasis caused by Trypanoreal extent of suramin resistance in T. soma evansi (surra) affects domestic live- evunsi since cases of suramin resistance are stock in countries from South America only infrequently reported in the literature through North Africa to the Middle and Far (Mahmoud and Gray 1980). East of Asia (Mahmoud and Gray 1980). To make more effective use of the availThe trypanosome is transmitted by several able trypanocides and to limit the spread of species of blood-sucking dipterous flies resistance to suramin, there is an urgent which makes controlling the disease by in- need for a method to detect, at an early secticide spraying impracticable. The only stage, the emergence of T. evunsi stocks effective method of dealing with sun-a is the resistant to suramin, particularly in counuse of chemotherapy. This depends on only tries where suramin is used intensively. four drugs: suramin, quinapyramine sul- The existing method of screening T. evunsi fate, isometamidium chloride, and dimina- stocks for drug resistance depends on obzene aceturate (Boid et al. 1985). In prac- serving the response of T. evunsi-infected tice, suramin is the drug used for treatment laboratory rodents to different amounts of of T. evunsi-infected animals. Suramin has the drug. Such a method is labour intenbeen in constant use as a trypanocide since sive, slow, and expensive. In order to im1925 (Williamson 1976) and reports of prove the existing methods, we looked for a suramin-resistant strains of T. evunsi are in- correlation between the isoenzyme banding creasing. Such strains have been reported patterns of 20 enzymes and suramin sensifrom Sudan (Leach 1961; Luckins et al. tivity in 29 stocks of T. evunsi isolated in 1979; Abebe et al. 1983), India (Gill 1971), North Africa and Indonesia. In this paper Russia (Petrovskii and Khamiev 1974), and we present evidence for a relationship between the suramin resistance of T. evansi stocks and the presence of a type VII isoenzyme (Gibson et al. 1980; Boid 1988) of r Present address: Balitvet, JaIan R. E. Martadinata malic enzyme. 32, P. 0. Box 52, Bogor, Java, Indonesia. INTRODUCTION
317 0014-4894/89$3.00 Copyright All riahts
0 1989 by Academic Press, Inc. of reoroduction in anv form reserved.
318
BOID,JONES, AND PAYNE MATERIALSAND
METHODS
Suramin. Suramin B. P. (Antrypol, ICI Ltd., Wilmslow, Cheshire) was used in this study as a sterile white powder. Solutions for injection were freshly prepared by dissolving the required amount of the drug in sterile distilled water at room temperature. Laboratory animals. Random-bred female albino CFl mice weighing 25 to 30 g were obtained from the
Centre for Laboratory Animals, University of Edinburgh. Ttypanosomes. AU trypanosome stocks used in this study were considered to represent examples of T. brucei evansi according to Hoare (1972). Trypanosome stocks were originally isolated from Egypt (1), Sudan (15), and Indonesia (13). Isolation details of the stocks are shown in Table I and Fig. 1. All trypanosome populations were derived from a single cloned
TABLE I T. evansi Stocks: Origin, Degree of Resistance to Suramin, and Malic Enzyme Type
Isolation details Stock no.
Host
Town
Country
Year
Group 1 1. T. evansi stock with type 1 suramin resistance and type II malic enzyme isoenzymes 1 Camel Soba Sudan Camel Kassala Sudan 2 Camel Kassala Sudan 3 Kassala Sudan 4 Camel Bovine ? Bali 5 Minahasa N. Sulawesi Bovine 6 Bovine Bolamongondo N. Sulawesi 7 Mlangi 8 Bovine E. Java Bovine Bogor W. Java 9 Telaga Mumi W. Java 10 Bovine Buffalo Gowa S. Sulawesi 11 Mlangi Buffalo E. Java 12 Buffalo Pekalongan C. Java 13 Ciawi W. Java 14 Buffalo Buffalo Bogor W. Java 15 Buffalo Bekasi W. Java 16 W. Java Buffalo Bogor 17
1977 Jan. 1977 1977 1977 1978 1984 1982 1983 1983 1983 1984 1983 1982 1984 1982 1984 1983
Group 2 2. T. evnnsi stock with type 2 suramin resistance and type II malic enzyme isoenzymes Shambat Sudan Camel 18 clone Shambat Sudan Camel 18 clone Sudan Camel Urn Hagar 19 clone Camel Urn Hagar Sudan 19 clone Camel Urn Gegeim Sudan 20 clone Urn Gegeim Sudan 20 clone Camel Sudan Camel Urn Gegeim 21 clone Urn Gegeim Sudan 21 clone Camel
Dec. Dec. Mar. Mar. Mar. Mar. Mar. Mar.
1976 1976 1977 1977 1977 1977 1977 1977
Oct. Oct. Oct. Apr. Apr. Jan. Jan.
1978 1978 1978 I978 1978 1977 1977 ?
Group 3 3. T. evansi stock with type 3 suramin resistance and type VII malic enzyme isoenzymes Soba Sudan Camel 22 Camel Soba Sudan 23 Camel Soba Sudan 24 Sudan 25 Camel Hadaliya 26 Camel Hadaliya Sudan 27 Camel Aroma Sudan 28 Camel Aroma Sudan Camel Cairo 29 Egypt
T.
evansi:
SURAMIN
30’
25’ I
SENSITIVITY
35’
EGYPT
A. FL LIBYA r
EHAD
SUDAN
___------Tsetse
Area
FIG. 1. Locations in Kassala Province, Sudan from which stocks of T. evansi were isolated.
population of each stock prepared by the method of Gardiner et al. (1980). A second cloned population was prepared from each of the stocks numbered 18, 19,20, and 21. Testing for drug sensitivity. Before use, stabilate material of each clone was expanded in mice which were bled at peak parasitaemia (4-6 days after infection) and the trypanosomes were separated from host erythrocytes by ion-exchange chromatography (Lanham and Godfrey 1970).
Column-separated trypanosomes of each stock were washed twice with PBSG, counted using a haemocytometer, and diluted with PBSG at pH 8.0 to give a final concentration of 5 X lo6 trypanosomeslml. Groups of five mice were used for each trypanosome stock at each suramin dose level. AU mice were inoculated intraperitoneally with 1 x lo6 trypanosomes. All living mice were examined for parasites daily, using wet films prepared from tail blood, for a period of 31 days after infection.
320
BOID, JONES, AND PAYNE
Twenty-four hours after infection, four mice from each group were injected intraperitoneally with 0.2 ml suramin at dose rates of 4,8, 16,32, or 64 mg/kg. The fifth mouse from each group acted as a control for that group and did not receive suramin.
stocks 18 to 21 inclusive were considered to be resistant to 4 mg/kg of suramin; parasitaemias in the mice receiving this dose were similar to those in control mice 4-8 days after treatment. Treatment of mice infected Extraction of malic enzyme (ME) (EC 1 .I .1.40) from trypanosomes. Trypanosomes representing each stock with any one of these four T. evansi stocks employed in the present study were separated from with suramin at dose rates of either 8 or 16 infected mouse blood by ion-exchange chromatography (Lanham and Godfrey 1970). The soluble en- mg/kg also did not eliminate the infection; zymes, including ME, were extracted from these rep- some of the mice developed sporadic pararesentative trypanosome populations and stored as sitaemias of between ~1 and lOO/HPF dur2&l beads in liquid nitrogen as described by Gibson ing the 31-day observation period. Mice inet al. (1978). fected with any one of these four stocks and Electrophoretic separation of malic enzyme. The electrophoretic mobility of ME was examined using then treated with suramin at a dose of 32 horizontal thin-layer starch gel electrophoresis using mg/kg became aparasitaemic 24-48 hr after the method of Wraxall and Culliford (1968) in 0.7- treatment and remained so for the rest of mm-thick gels using a starch concentration of 10.6% the 31-day observation period. New cloned w/v. Electrophoresis was carried out at 300 V and at a populations were prepared from each of temperature of 8°C for 3 hr using a 0.2 M phosphate these four stocks and the experiment was buffer, pH 7.4, system (Boid 1988). Malic enzyme bands were visualized with a stain repeated with one example of each new solution containing 10 ml 0.5 M Tris/HCl buffer, pH clone; similar results were obtained. 7.4; 2 ml 0.1 M L-malate, pH 7.0; 2 ml MTT (4 mg/mI); Group 3: Mice infected with T. evansi 100 pl meldola blue (6 mg/ml); 1 ml NADP (10 mg/ml); stocks 22 to 29 inclusive differed markedly mixed with 10 ml warm agar solution (70 mg/ml). Gels in their reaction to suramin when compared were incubated in the dark at 37°C for 30 min. The nomenclature used for the isoenzyme banding patterns with the other stocks tested in groups 1 and of malic enzyme is that used by Gibson et al. (1980). 2. Mice infected with any one of the eight stocks and then treated with suramin at all RESULTS doses of up to 64 mg/kg all reached parasitaemias of lOO/HPF within 3 to 5 days after Suramin sensitivity of T. evansi stocks treatment. Control mice. Trypanosomes appeared in all mice 24 hr after infection and parasi- Isoenzymes taemias increased progressively until 100 T. evansi stocks 1 to 21 inclusive were trypanosomes/HPF were present 4 to 8 found to have type II isoenzyme banding days after infection. Control mice generally patterns for malic enzyme. The eight other died with a fulminating parasitaemia 8 to 18 T. evansi stocks, 22 to 29 inclusive, had a days after infection. type VII isoenzyme banding pattern for Suramin-treated mice. These results are malic enzyme (Table I). summarised in Table I. Trypanosome The two enzyme banding patterns are ilstocks were categorized into groups 1, 2, lustrated diagrammatically in Fig. 2. Bands and 3 according to their degree of resis- b and c are present in the type II pattern but are absent from the type VII pattern. The tance to suramin. Group 1: Mice infected with T. evansi absence of any significant enzymic activity stocks 1 to 17 inclusive were all trypano- at positions b and c was confirmed by loadsome negative 24 hr after injection of ing gels with fivefold concentrated samples. suramin at a dose of 4 mg/kg. Thereafter no DISCUSSION trypanosomes were seen in the blood during the observation period of 31 days. The 29 trypanosome stocks examined in Group 2: Mice infected with T. evansi this work showed a range of sensitivities to
T. evansi: SURAMIN SENSITIVITY Type II
Type VII +
Cba-
-
-a
-
origin
FIG. 2. Diagram of the type II and type VII enzyme banding patterns of malic enzyme from stocks of T. evansi. n , strong enzyme activity. Each enzyme band is designated by a lowercase letter.
the trypanocide suramin. Trypanosome stocks belonging to group 1 were sensitive to the normal dose of suramin (4 mg/kg) required to clear suramin-sensitive stocks of T. evansi from infected mouse blood (Abebe et al. 1983). Stocks ascribed to group 2 were less sensitive than group 1 stocks and required suramin at dose rates of between 16 and 32 mg/kg to clear them from mouse blood. Group 3 trypanosome stocks were highly resistant to suramin at dose rates of up to 64 mg/kg. Correlation of the suramin resistance of the previous groups with the isoenzyme banding patterns of malic enzyme showed that all T. evansi stocks which had some degree of suramin sensitivity (groups 1 and 2) exhibited a type II malic enzyme banding pattern (see Table I). Group 3 stocks, which were highly resistant to suramin, had a type VII enzyme banding pattern. Three isoenzyme banding patterns of malic enzyme, types II, VII, and XXIV, have been reported to occur in T. evansi (Gibson et al. 1980;Boid 1988).The type II pattern represents the most frequently encountered banding pattern for this enzyme in T. evunsi and has been reported in stocks isolated from Colombia, Nigeria, Kenya, Sudan, and Indonesia (Gibson et al. 1980, 1983; Boid 1988). Only one example of the
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type XXIV pattern has been reported (Gibson et al. 1983).The type VII malic enzyme banding pattern has only been reported from eight stocks of T. evunsi (Boid 1988). Two of the eight stocks were isolated at Aroma in 1977;two stocks were isolated at Hadaliya, some 50 km north of Aroma, in 1978; three stocks were isolated at Soba, some 300 km to the west, in 1978; and one stock was obtained from Cairo, Egypt (Fig. 1). The nomenclature used for the isoenzyme banding patterns of malic enzyme is consistent with that used by Gibson et al. (1980) for members of the subgenus Trypunozoon.
The results of this study suggest that stocks of T. evunsi which are found to have a type VII malic enzyme banding pattern may be highly suramin resistant (>64 mg/ kg). Previous workers have examined stocks of T. brucei and failed to establish a correlation between particular isoenzyme banding patterns (including malic enzyme) and resistance to the trypanocidal drugs trypamidium, melarsoprol, pentamidine, and berenil (Dukes 1984). Isoenzymically defined West African stocks were shown to be more sensitive to pentamidine and berenil than East African stocks. No experimental evidence has been published to explain the trypanocidal action of suramin on T. evunsi. The results of this study suggest that the trypanocidal action of suramin against T. evunsi may be due, at least in part, to the inhibition of one or more isoenzymes of malic enzyme. A preliminary investigation has been carried out on unpurified enzyme preparations from one suramin-sensitive T. evunsi (stock 2, Table I) and one suramin-resistant T. evunsi (stock 28, Table I). Under the conditions of the assay, 20 @4 suramin inhibited the activity of malic enzyme in the suraminsensitive stock by approximately twice as much as it inhibited the enzyme in the suramin-resistant stock (R. Boid, unpublished). The action of suramin on the isoen-
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BOID,JONES, AND PAYNE
zyme types of suramin is being further investigated. Of the T. evansi stocks examined in this work, stocks that were highly resistant to suramin were only found in Northeast Africa and not in Indonesia. This probably reflects the constant exposure of trypanosome-infected animals to chemotherapeutic treatment in these countries, which has undoubtedly led to the spread of drugresistant strains. Despite this, there is no detailed information on drug resistance in T. evansi (Schillinger 1984)and there are no simple tests to determine the drug sensitivity of trypanosome stocks isolated in the field. The identification of malic enzyme as a marker for suramin resistance in stocks of T. evansi should eventually allow the development of an enzyme-linked immunosorbent assay to detect suramin-resistant stocks of T. evansi. The availability of such a test would let the veterinarian select alternative chemotherapeutic regimes to control infection and limit the spread of suramin resistance. ACKNOWLEDGMENTS The authors thank Dr. P. Stevenson, Mr. A. Day,
BPPH, for isolating the trypanosome stocks in Indonesia, and Dr. A. J. Wilson and Dr. H. R. Pumomo, Director, BPPH, for providing facilities in Indonesia. The assistance of the Dean of the Faculty of Veterinary Science, University of Khartoum, and the cooperation of the staff of the Sudan Ministry of Agriculture and Natural Resources is gratefully acknowledged. Professor Dr. F. Hiirchner, Institut fur parasitologie und tropenveterinarmedizin, Frae Universitat Berlin, kindly provided the Egyptian stock of T. evansi. Financial assistance was provided by the Overseas Development Administration of the United Kingdom.
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