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Differences in cloning and sub-cloning success rates in four stocks of Trypanosoma evansi and variation in suramin resistance of the clones
veterinary parasitology ELSEVIER
Veterinary Parasitology 60 (1995) 213-220
Differences in cloning and sub-cloning success rates in four stocks of Trypanosoma evansi and variation in suramin resistance of the clones M.W. Mutugi a,*, R. Boid b, A.G. Luckins b Kenya Trypanosoraiasis Research Institute (KETRI), P.O. Box 362, Kikuyu, Kenya b Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin, H25 9RG, UK Accepted 22 December 1994
Abstract Four Trypanosomaevansi stocks with sensitivity to suramin in mice ranging from 0.05 to 160 mg kg-~ were cloned and sub-cloned and the sensitivity of the clones determined. The results suggest that it is easier to clone and sub-clone trypanosome stocks which are sensitive to suramin than those that are resistant to the action of the drug. The clones obtained from the four stocks had sensitivities to suramin which were similar to or different from the parent stocks. These results are important in view of the development of resistance for, in the presence of suramin, these resistant yet heterogeneous populations would provide the material from which selective processes could operate. These observations also suggest that the maintenance and spread of suramin-resistant trypanosomes might be curtailed by their comparative inability to establish themselves in a new host. Keywords: Trypanosoma evansi; Suramin; Resistance; Clones; Sub-clones
I. Introduction Suramin was developed at the beginning of this century, and became available in 1920 for the control of parasites of the Trypanozoon subgenus (Knowles, 1925). Together with quinapyramine, this drug is important for the control of Trypanosoma evansi infections especially in camels since other trypanocidal drugs such as Berenil and Samorin are not well tolerated. There have been instances, however, whereby the effectiveness of this drug has been reduced by the development of resistance in
* Corresponding author at: 12 Shottery Road, Greystone Park, Harare, Zimbabwe. 0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 7 8 7 - 3
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trypanosome populations. Such reports have been reported in infected buffaloes in Indonesia (Kraneveld and Mansjoer, 1941), infected ponies in India (Gill, 1971) as well as infected camels in Sudan (Evans, 1946; Leach, 1961; Luckins et al., 1979; Boid et al., 1985, 1989), the former USSR (Petrovskii and Khamiev, 1974) and in Kenya (Gitatha, 1980; Schillinger et al., 1985). Both in vivo and in vitro studies indicate that suramin resistance is stable and thus trypanosomes that have acquired this trait maintain it in the absence of the drug (Morgenroth and Freud, 1924; Amrein and Fulton, 1959; Zweygarth et al., 1991; Mutugi et al., 1994). There have also been instances where stocks of T. evansi have been shown to be resistant to both quinapyramine and suramin (Mahmoud and Osman, 1979; Schillinger et al., 1985). The extent of T. evansi resistance to trypanocidal drugs recommended for its control is not known, however. The evidence for mechanisms underlying the development of resistance to trypanocidal drugs suggests that a change in genetic constitution, mutation and selection of that change play a major role. Thus, "appearance of resistant mutants is a spontaneous sporadic occurrence.., exposure (to a drug) only selects m u t a n t s . . . " (Schnitzer and Hawking, 1966). The mutational theory for the origin of drug resistance is supported by the observation that, even when a trypanosome population is derived from a single parasite by cloning, the trypanosomes comprising the population differ in their sensitivity to a particular trypanocidal drug. For example, in a strain of Trypanosoma congolense with an overall Berenil sensitivity of 35.7 mg kg-t and Samorin sensitivity of 3.6 mg kg -~, clones derived from the population were shown to have sensitivities to Berenil ranging from 5.1 to 21 mg kg-1 and Samorin sensitivities ranging from of 1.5 to 5.1 mg kg -1 (Peregrine et al., 1990). This implies that, although the original cloned organisms possess a particular level of drug sensitivity, as multiplication takes place and the population increases, mutants arise whose drug sensitivities differ from their parents. When a trypanocidal drug is introduced into such a population, trypanosomes which are resistant to the drug at the dose level used survive, while those that are sensitive are destroyed. The objective of this work was to clone and sub-clone four T. evansi stocks and determine the suramin sensitivities of the derived clones. This was to investigate factors which influence the development and maintenance of suramin-resistant populations.
2. Materials and methods 2.1. Trypanosomes
The trypanosome stocks used in this work were isolated from naturally infected camels from Kenya and Sudan. KETRI 2454 and KETRI 2476 were isolated from Marsabit in 1979 and Ngurunit in 1980, respectively, in Kenya, and were resistant to suramin, in mice, at a dose rate of 0.005 mg kg -1. KETRI 2455 was isolated from Galana, Kenya, in 1979 and is resistant to suramin in mice at a dose rate of 10 mg kg -1 , while KETRI 3136 was isolated in Kassala, Sudan, in 1977 and was shown to be resistant to the action of suramin in mice at a dose rate of 160 mg kg -1. The suramin sensitivities of these stocks were established in a related study (Mutugi, 1993) where
M. W. Mutugi et a l l Veterinary Parasitology 60 (1995) 213-220
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resistance to a particular trypanocidal drug was a dose which did not cure 80% of infected mice. Thus, after drug administration, infected mice remained parasitaemic or reappearance of parasites was observed within 60 days. 2.2. Mice The mice used in this work were male Swiss white albino weighing between 20 and 25 g from the KETRI disease-free animal breeding unit. 2.3. Clone preparation Cloned populations were prepared from four trypanosome stocks, KETRI 2454, 2455, 2476 and 3136, using a modified method of Van Meirvenne et al. (1975) and Gardiner et al. (1980). In this method, a buffer prepared by mixing phosphate-saline-glucose buffer (PSG) with guinea pig serum in a ratio of 1:1 was used for all dilutions. Blood from a mouse with a high parasitaemia was diluted with the buffer until the parasitaemia was approximately one parasite in five fields (magnification ×400; Leitz Wetzlar SM-Lux, Germany). A 10 ml aliquot of buffer was put into the cavity of a haemocytometer (Neubauer Improved Double Ruling Superior, Germany) in order to maintain a moisturised environment. A 10 ml volume of the infected blood diluted with buffer was then put on a coverslip and the coverslip inverted over the cavity slide of the haemocytometer avoiding touching the buffer present in the cavity. The sample was then examined microscopically (X 400 magnification) and if the parasitaemia was too high, an appropriate dilution was made to decrease the number of trypanosomes to one per preparation. When a single trypanosome was observed in the sample, a second person confn-rned its presence after which the drop containing the trypanosome was mixed with 0.1 ml of buffer contained in a 1 ml syringe. The trypanosome in buffer was then inoculated intraperitoneally into a mouse which had been immunosuppressed 24 h earlier by a single intraperitoneal administration of cyclophosphamide monohydrate (Endoxana; Koch-Light Laboratories, UK) at 300 mg kg-1 body weight. The cloning procedure was repeated until ten mice were inoculated for each isolate under investigation. The mice were monitored daily for parasitaemia by wet blood analysis. Any development of parasitaemia in the inoculated mice represented cloned trypanosome populations. If none of the mice inoculated with single trypanosomes became parasitaemic, the experiment was repeated. Cloning success for each of the stocks was calculated as the proportion of successful infections out of all mice inoculated with a cloned parasite. The same cloning procedure was repeated using the most resistant clone from each of the four stocks, and thus sub-clones were obtained and sub-cloning success rates calculated. 2.4. Determination of suramin sensitivities The lowest suramin dose to which the trypanosome stocks and the clones were sensitive was determined by a titration experiment using suramin at dose rates as shown
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Table 1 Suramin doses used to determine the sensitivities of four T. evansi stocks and clones derived from them Stock KETRI no.
Suramin doses used (mg kg -1 )
2454, 2476 2455 3136
0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 1 1,2, 4,5,10,15,20 10,20,40,60,80,100,120,140, 160
in Table 1. These dose ranges were chosen in view of the known sensitivity of the isolates to suramin as indicated in previous experiments. For each isolate and drug dose level, six mice were infected intraperitoneally with 1 × 10 6 trypanosomes and five of them treated with suramin 24 h later. The sixth mouse was used as an untreated control. All mice were screened daily for parasites for a period of 60 days by microscopic examination of tail blood.
3. Results The parent stocks, KETRI 2454 and 2476, were resistant to suramin at a dose level of 0.005 mg kg-1 body weight but were sensitive at dose rates of 0.01 mg kg-1 body weight and above; KETRI 2455 was resistant to suramin at a dose of 10 mg kg-~ but was sensitive to 15 mg kg-~ and above, while KETRI 3136 was resistant to 160 mg kg -1 " A cloning success rate of 60% was achieved using KETRI 2454 and KETRI 2476, but with KETRI 2455 and KETRI 3136 the success rates were much lower at 8.3% and 1.7%, respectively (Table 2). The different clones produced from the four trypanosome stocks had suramin sensitivities ranging from 0.005 to 120 mg kg -1 (Table 3). For KETRI 2454 and 2476, one clone from each stock had a similar sensitivity to the parents (0.005 mg kg - ~ ) but the other five clones had suramin sensitivities greater than the parent stocks. The clone of KETRI 2455 had a sensitivity of 2 mg kg -~, which was lower than the sensitivity of the parent stock (10 mg k g - ~). The four clones of KETRI 3136 had suramin sensitivities ranging from 60 to 120 mg kg -1 , which are lower than the sensitivity of the parent stock (160 mg k g - ~).
Table 2 Cloning success rates of four T. evansi stocks, KETRI 2454, 2455, 2476 and 3136 KETRI no.
Resistance to suramin (rag kg- i )
No. of clones established
No. of cloning attempts
Cloning success (%)
2454 2455 2476 3136
0.005 10 0.005 160
6 1 6 4
10 60 10 48
60 1.7 60 8.3
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Table 3 Suramin sensitivitiesof clones derived from T. evansi stocks, KETRI 2454, 2455, 2476 and 3136 Isola~ KETRIno.
Cloneno.
Suramin~sistance (mgkg -j )
2454
1 2 3 4 5 6 1 1 2 3 4 5 6 1 2 3 4
0.2 0.1 0.1 0.05 0.2 0.005 2 0.2 0.005 0.05 0.05 0.1 0.1 80 80 120 60
2455 2476
3136
The clones that exhibited the highest level of resistance to suramin were Clone 1 for KETRI 2454, 2455 and 2476 and Clone 3 for KETRI 3136. These clones were further cloned and sub-cloning success rates for KETRI 2454 was 80%, 50% for KETRI 2476, and 0% and 30% for KETRI 2455 and 3136, respectively (Table 4).
4. D i s c u s s i o n
There was a wide range of cloning success rates with the four T. evansi stocks examined. Barry et al. (1979) reported cloning success rates of 70% and 50% in Trypanosoma b. rhodesiense and Trypanosoma b. brucei respectively. These workers reported different cloning success rates in different species as compared to the results in this study where the success rates of cloning differed between different stocks of the same species. A greater success rate was obtained with the suramin-sensitive stocks of T. eoansi ( K E T R I 2454 and 2476) compared with the resistant stocks (KETRI 2455 and
Table 4 Subcloningsuccess rates of four T. evansi stocks, KETRI 2454, 2455, 2476 and 3136 KETRI no.
Resistance to suramin (mg kg- i )
No. of subclones established
No. of subcloning attempts
Subcloning success (%)
2454 Clone 1 2455 Clone 1 2476 Clone 1 3136 Clone 3
0.2 2 0.2 120
8 0 5 3
10 10 10 10
80 0 50 30
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3136). To a lesser extent, a similar trend was observed in sub-cloning, thus greater cloning success rates were observed when the clones of the suramin-sensitive stocks were sub-cloned. This success, however, was not directly related to the degree of suramin resistance, as the lowest success rate was obtained with KETRI 2455 and its clone which were resistant to suramin at dose rates of 10 and 2 mg k g - f body weight, respectively, and not with KETRI 3136 and its clone, resistant at dose rates of 160 and 120 mg kg-~ body weight respectively. Cloning success is determined by the ability of a single parasite to withstand the host's defence mechanisms and establish an infection. This may be related to some innate characteristics of the drug-sensitive trypanosome, such as a fast growth rate or high turnover of variable antigen types, which enables it to proliferate quickly in order to evade host-defence mechanisms. Although only four trypanosome stocks were investigated, the reduced cloning success rates obtained with the suramin-resistant trypanosomes suggests that they might establish less readily than sensitive stocks in vertebrate hosts. This agrees with the observation of Cantrell (1956) who studied the behaviour of drug-sensitive and resistant T. equiperdum parasites in mixed infections, and concluded that " m o s t drug resistant strains are less well adapted to life in the absence of the drug than their unmodified parents". Since clones from a single trypanosome stock showed a range of suramin sensitivities, field isolates are probably mixed populations as far as suramin sensitivity is concerned. This is the first time that T. evansi stocks have been shown to be heterogeneous populations with respect to drug sensitivity. Similar findings have been reported in other salivarian trypanosomes such as T. congolense (Peregrine et al., 1990). In that study, nine clones of the parent isolate had a spectrum of resistance to isometamidium chloride ranging from 1.5 to 5.1 mg kg-~ body weight while the range of resistance to diminazene aceturate was from 5.1 to 21 mg kg -1 . All of the clones of KETRI 2455 and 3136 and those investigated by Peregrine et al. (1990) had sensitivities to suramin and diminazene aceturate, respectively, which were lower than that of the parent isolate (35.7 mg kg-~). Similarly, the present observation with T. evansi KETRI 2454 and 2476, and the study on T. congolense using isometamidium (Peregrine et al., 1990), showed that some clones were more resistant than the parent stock. All other clones, however, had sensitivities which were lower than the parent stocks. Walker (1964) determined that ten resistant trypanosomes in a million are enough to give an isolate a resistant phenotype. One can thus assume that a heterogeneous stock must have at least this proportion of parasites, whose suramin sensitivity is the same as that of the parent isolate. If this assumption is correct, then there must have been at least this proportion of clones which gave identity to the stocks used in this experiment and, it is likely that they may have been among the ones inoculated into mice resulting in the failed cloning attempts. This would thus suggest that the resistant clones are less adapted to withstand the hostility of the host's defences than their sensitive counterparts as indicated by failure to establish infection. Hence, in the study of Peregrine et al. (1990), the clone which was more resistant to isometamidium chloride than the parent stock might thus have consisted of less than 0.001% of the total population. This argument would explain why most clones in this work and in the study of Peregrine et al. (1990) were of lower drug sensitivities compared to their parent clones.
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In conclusion, this study has shown that four stocks of T. evansi consist of trypanosomes with sensitivities to suramin which are either the same as or lower than that of the parent stocks. Thus, with or without suramin, trypanosomes exhibited a range of suramin sensitivities and introduction of the drug would only select for the more resistant parasites. The success of cloning T. evansi in this study varied between stocks and clones of different suramin sensitivities and may have been related to the level of suramin sensitivity. The observation that drug-resistant trypanosomes were more difficult to clone and sub-clone compared to drug-sensitive ones suggests that a single resistant trypanosome is unable to survive in a mouse as readily as a sensitive one. Thus, cloning may be hindered by some inability of the drug-resistant parasites to establish infections as compared with drug-sensitive organisms. Although mice were used in this study, the findings observed may have implications in the establishment and maintenance of suramin resistance in T. evansi infecting camels under field conditions.
Acknowledgements This work was carried out as part of a Ph.D. programme at the University of Edinburgh by the first author funded by the Overseas Development Administration of the UK and Kenya Trypanosomiasis Research Institute (KETRI), and is published with the kind permission of the Director KETRI.
References Amrein, Y.U. and Fulton, J.D., 1959. Attempts to transfer drug resistance to trypanosomes in vivo. J. Protozool., 6: 120-122. Barry, J.D., Le Ray, D. and Herbert, W.J., 1979. Infectivity and virulence of Trypanosoma (Trypanozoon) brucei in mice. IV. Dissociation of virulence and variable antigen types in relation to pleomorphisms. J. Comp. Pathol., 89: 465-470. Boid, R., Jones, T.W. and Luckins, A.G., 1985. The camel in health and disease. 3. Protozoal diseases of camels. Br. Vet. J., 141: 87-104. Boid, R., Jones, T.W. and Payne, R.C., 1989. Malic enzyme type VII isoenzyme as an indicator of suramin resistance in Trypanosoma evansi. Exp. Parasitol., 69: 317-323. Cantrell, W., 1956. Behaviour of mixtures of oxophenarsine-resistant and unmodified strains of Trypanosoma equiperdum in the rat. Exp. Parasitol., 5: 178-190. Evans, J.T.R., 1946. Annual Report of the Veterinary Department of the Sudan Government (cited by Gau-Rutter, T.E., 1967. Diseases of camels. Vet. Bull., 37:611-618). Gardiner, P.R., Jones, T.W. and Cunningham, I., 1980. Antigen analysis by immunofluorescence of in vitro produced metacyclics of Trypanosoma brucei and their infections in mice. J. Protozool., 27: 316-320. Gill, B.S., 1971. Drug resistance in Trypanosoma evansi. Trop. Anim. Health Prod., 3: 195-198. Gitatha, S.K., 1980. Report of Trypanosoma evansi stabilates isolated from camels in Kenya. Proceedings of 1st Annual Medical Scientific Conference of KEMRI/KETRI, Nairobi, Kenya, February 1980, pp. 183-186. Knowles, R.H., 1925. Treatment of camels affected with T. congolense with 'Bayer 205' and further observations on the formol-gel test. J. Comp. Pathol., 40:118-193. Kraneveld, F.C. and Mansjoer, M., 1941. Proeven over vervangproduction voor Naganol Bayer en Arsocoll. I. Nagonal Specia. Ned. Indische BI. Diergeneeskd., 53: 465-475.
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Leach, T.H., 1961. Observations on the lreatment of Trypanosoma evansi infection in camels. J. Comp. Pathol., 71: 109-117. Luckins, A.G., Boid, R., Rae, P.F., Mahmoud, M.M. and El Malik, A.R., 1979. Serodiagnosis of infection of Trypanasoma evansi in camels in the Sudan. Trop. Anita. Health Prod., 1 l: 1-17. Mahmoud, M.M. and Osman, O.M., 1979. Trypanosomiasis of Sudan camels. International Foundation for Science (IFS), Report No. 6, pp. 431-439. Morgenroth, J. and Freud, R., 1924. Uber die Wirkungsweise yon 'Bayer 205' bei und experimentellen Trypanosomen-Infektion der Maus. Klin. Wochenschr., 2: 53-55. Mutugi, M.W., 1993. Studies on suramin resistance in Kenyan stocks of Trypanosoma evansi. Ph.D. Thesis, University of Edinburgh. Mutugi, M.W., Boid, R. and Luckins, A.G., 1994. Experimental induction of suramin-resistance in cloned and uncloned stocks of Trypanosoma evansi using immunosuppressed and immunocompetent mice. Trop. Med. Parasitol., 45: 232-236. Peregrine, A.S., Knowles, G., Ibitayo, A.I., Moloo, S.K. and Murphy, N.B., 1990. Variation in resistance to isometamidium chloride and diminazene aceturate by clones derived from a stock of Trypanosoma congolense. Parasitology, 102: 92-100. Petrovskii, V.V. and Khamiev, S.K.H., 1974. Cited by Boid, R., Jones, T.W. and Luckins, A.G., 1985. The camel in health and disease. 3. Protozoal diseases of camels. Br. Vet. J., 141: 87-104. Schillinger, D., Maloo, S.H. and Rottcher, D., 1985. Trypanosoma evansi edemy in dromedary herds of Kenya --drug resistance and chemotherapy. Camel Newsl., 1: 13-16. Schnitzer, R.J. and Hawking, F. (Editors), 1966. Experimental Chemotherapy, Vol. I. Academic Press, New York, pp. 405-406. Van Meirvenne, N., Janssens, P.G. and Magnus, E., 1975. Antigenic variation in syringe passaged populations of Trypanosoma (Trypanozoon) brucei. I. Rationalisation of the experimental approach. Ann. Soc. Belg. Med. Trop., 55: 1-23. Walker, P.J., 1964. Reproduction and heredity in trypanosomes. Int. Rev. Cytol., 17: 51-98. Zweygarth, E., Kaminsky, R. and Webster, P., 1991. Trypanosoma brucei evansi: dyskinetoplasia and loss of infectivity after long-term in vitro cultivation. Acta Trop., 48: 95-99.
Veterinary Parasitology 60 (1995) 213-220
Differences in cloning and sub-cloning success rates in four stocks of Trypanosoma evansi and variation in suramin resistance of the clones M.W. Mutugi a,*, R. Boid b, A.G. Luckins b Kenya Trypanosoraiasis Research Institute (KETRI), P.O. Box 362, Kikuyu, Kenya b Centre for Tropical Veterinary Medicine, University of Edinburgh, Easter Bush, Roslin, H25 9RG, UK Accepted 22 December 1994
Abstract Four Trypanosomaevansi stocks with sensitivity to suramin in mice ranging from 0.05 to 160 mg kg-~ were cloned and sub-cloned and the sensitivity of the clones determined. The results suggest that it is easier to clone and sub-clone trypanosome stocks which are sensitive to suramin than those that are resistant to the action of the drug. The clones obtained from the four stocks had sensitivities to suramin which were similar to or different from the parent stocks. These results are important in view of the development of resistance for, in the presence of suramin, these resistant yet heterogeneous populations would provide the material from which selective processes could operate. These observations also suggest that the maintenance and spread of suramin-resistant trypanosomes might be curtailed by their comparative inability to establish themselves in a new host. Keywords: Trypanosoma evansi; Suramin; Resistance; Clones; Sub-clones
I. Introduction Suramin was developed at the beginning of this century, and became available in 1920 for the control of parasites of the Trypanozoon subgenus (Knowles, 1925). Together with quinapyramine, this drug is important for the control of Trypanosoma evansi infections especially in camels since other trypanocidal drugs such as Berenil and Samorin are not well tolerated. There have been instances, however, whereby the effectiveness of this drug has been reduced by the development of resistance in
* Corresponding author at: 12 Shottery Road, Greystone Park, Harare, Zimbabwe. 0304-4017/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0 3 0 4 - 4 0 1 7 ( 9 5 ) 0 0 7 8 7 - 3
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trypanosome populations. Such reports have been reported in infected buffaloes in Indonesia (Kraneveld and Mansjoer, 1941), infected ponies in India (Gill, 1971) as well as infected camels in Sudan (Evans, 1946; Leach, 1961; Luckins et al., 1979; Boid et al., 1985, 1989), the former USSR (Petrovskii and Khamiev, 1974) and in Kenya (Gitatha, 1980; Schillinger et al., 1985). Both in vivo and in vitro studies indicate that suramin resistance is stable and thus trypanosomes that have acquired this trait maintain it in the absence of the drug (Morgenroth and Freud, 1924; Amrein and Fulton, 1959; Zweygarth et al., 1991; Mutugi et al., 1994). There have also been instances where stocks of T. evansi have been shown to be resistant to both quinapyramine and suramin (Mahmoud and Osman, 1979; Schillinger et al., 1985). The extent of T. evansi resistance to trypanocidal drugs recommended for its control is not known, however. The evidence for mechanisms underlying the development of resistance to trypanocidal drugs suggests that a change in genetic constitution, mutation and selection of that change play a major role. Thus, "appearance of resistant mutants is a spontaneous sporadic occurrence.., exposure (to a drug) only selects m u t a n t s . . . " (Schnitzer and Hawking, 1966). The mutational theory for the origin of drug resistance is supported by the observation that, even when a trypanosome population is derived from a single parasite by cloning, the trypanosomes comprising the population differ in their sensitivity to a particular trypanocidal drug. For example, in a strain of Trypanosoma congolense with an overall Berenil sensitivity of 35.7 mg kg-t and Samorin sensitivity of 3.6 mg kg -~, clones derived from the population were shown to have sensitivities to Berenil ranging from 5.1 to 21 mg kg-1 and Samorin sensitivities ranging from of 1.5 to 5.1 mg kg -1 (Peregrine et al., 1990). This implies that, although the original cloned organisms possess a particular level of drug sensitivity, as multiplication takes place and the population increases, mutants arise whose drug sensitivities differ from their parents. When a trypanocidal drug is introduced into such a population, trypanosomes which are resistant to the drug at the dose level used survive, while those that are sensitive are destroyed. The objective of this work was to clone and sub-clone four T. evansi stocks and determine the suramin sensitivities of the derived clones. This was to investigate factors which influence the development and maintenance of suramin-resistant populations.
2. Materials and methods 2.1. Trypanosomes
The trypanosome stocks used in this work were isolated from naturally infected camels from Kenya and Sudan. KETRI 2454 and KETRI 2476 were isolated from Marsabit in 1979 and Ngurunit in 1980, respectively, in Kenya, and were resistant to suramin, in mice, at a dose rate of 0.005 mg kg -1. KETRI 2455 was isolated from Galana, Kenya, in 1979 and is resistant to suramin in mice at a dose rate of 10 mg kg -1 , while KETRI 3136 was isolated in Kassala, Sudan, in 1977 and was shown to be resistant to the action of suramin in mice at a dose rate of 160 mg kg -1. The suramin sensitivities of these stocks were established in a related study (Mutugi, 1993) where
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resistance to a particular trypanocidal drug was a dose which did not cure 80% of infected mice. Thus, after drug administration, infected mice remained parasitaemic or reappearance of parasites was observed within 60 days. 2.2. Mice The mice used in this work were male Swiss white albino weighing between 20 and 25 g from the KETRI disease-free animal breeding unit. 2.3. Clone preparation Cloned populations were prepared from four trypanosome stocks, KETRI 2454, 2455, 2476 and 3136, using a modified method of Van Meirvenne et al. (1975) and Gardiner et al. (1980). In this method, a buffer prepared by mixing phosphate-saline-glucose buffer (PSG) with guinea pig serum in a ratio of 1:1 was used for all dilutions. Blood from a mouse with a high parasitaemia was diluted with the buffer until the parasitaemia was approximately one parasite in five fields (magnification ×400; Leitz Wetzlar SM-Lux, Germany). A 10 ml aliquot of buffer was put into the cavity of a haemocytometer (Neubauer Improved Double Ruling Superior, Germany) in order to maintain a moisturised environment. A 10 ml volume of the infected blood diluted with buffer was then put on a coverslip and the coverslip inverted over the cavity slide of the haemocytometer avoiding touching the buffer present in the cavity. The sample was then examined microscopically (X 400 magnification) and if the parasitaemia was too high, an appropriate dilution was made to decrease the number of trypanosomes to one per preparation. When a single trypanosome was observed in the sample, a second person confn-rned its presence after which the drop containing the trypanosome was mixed with 0.1 ml of buffer contained in a 1 ml syringe. The trypanosome in buffer was then inoculated intraperitoneally into a mouse which had been immunosuppressed 24 h earlier by a single intraperitoneal administration of cyclophosphamide monohydrate (Endoxana; Koch-Light Laboratories, UK) at 300 mg kg-1 body weight. The cloning procedure was repeated until ten mice were inoculated for each isolate under investigation. The mice were monitored daily for parasitaemia by wet blood analysis. Any development of parasitaemia in the inoculated mice represented cloned trypanosome populations. If none of the mice inoculated with single trypanosomes became parasitaemic, the experiment was repeated. Cloning success for each of the stocks was calculated as the proportion of successful infections out of all mice inoculated with a cloned parasite. The same cloning procedure was repeated using the most resistant clone from each of the four stocks, and thus sub-clones were obtained and sub-cloning success rates calculated. 2.4. Determination of suramin sensitivities The lowest suramin dose to which the trypanosome stocks and the clones were sensitive was determined by a titration experiment using suramin at dose rates as shown
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Table 1 Suramin doses used to determine the sensitivities of four T. evansi stocks and clones derived from them Stock KETRI no.
Suramin doses used (mg kg -1 )
2454, 2476 2455 3136
0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 1 1,2, 4,5,10,15,20 10,20,40,60,80,100,120,140, 160
in Table 1. These dose ranges were chosen in view of the known sensitivity of the isolates to suramin as indicated in previous experiments. For each isolate and drug dose level, six mice were infected intraperitoneally with 1 × 10 6 trypanosomes and five of them treated with suramin 24 h later. The sixth mouse was used as an untreated control. All mice were screened daily for parasites for a period of 60 days by microscopic examination of tail blood.
3. Results The parent stocks, KETRI 2454 and 2476, were resistant to suramin at a dose level of 0.005 mg kg-1 body weight but were sensitive at dose rates of 0.01 mg kg-1 body weight and above; KETRI 2455 was resistant to suramin at a dose of 10 mg kg-~ but was sensitive to 15 mg kg-~ and above, while KETRI 3136 was resistant to 160 mg kg -1 " A cloning success rate of 60% was achieved using KETRI 2454 and KETRI 2476, but with KETRI 2455 and KETRI 3136 the success rates were much lower at 8.3% and 1.7%, respectively (Table 2). The different clones produced from the four trypanosome stocks had suramin sensitivities ranging from 0.005 to 120 mg kg -1 (Table 3). For KETRI 2454 and 2476, one clone from each stock had a similar sensitivity to the parents (0.005 mg kg - ~ ) but the other five clones had suramin sensitivities greater than the parent stocks. The clone of KETRI 2455 had a sensitivity of 2 mg kg -~, which was lower than the sensitivity of the parent stock (10 mg k g - ~). The four clones of KETRI 3136 had suramin sensitivities ranging from 60 to 120 mg kg -1 , which are lower than the sensitivity of the parent stock (160 mg k g - ~).
Table 2 Cloning success rates of four T. evansi stocks, KETRI 2454, 2455, 2476 and 3136 KETRI no.
Resistance to suramin (rag kg- i )
No. of clones established
No. of cloning attempts
Cloning success (%)
2454 2455 2476 3136
0.005 10 0.005 160
6 1 6 4
10 60 10 48
60 1.7 60 8.3
M.W. Mutugi et al./ Veterinary Parasitology 60 (1995) 213-220
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Table 3 Suramin sensitivitiesof clones derived from T. evansi stocks, KETRI 2454, 2455, 2476 and 3136 Isola~ KETRIno.
Cloneno.
Suramin~sistance (mgkg -j )
2454
1 2 3 4 5 6 1 1 2 3 4 5 6 1 2 3 4
0.2 0.1 0.1 0.05 0.2 0.005 2 0.2 0.005 0.05 0.05 0.1 0.1 80 80 120 60
2455 2476
3136
The clones that exhibited the highest level of resistance to suramin were Clone 1 for KETRI 2454, 2455 and 2476 and Clone 3 for KETRI 3136. These clones were further cloned and sub-cloning success rates for KETRI 2454 was 80%, 50% for KETRI 2476, and 0% and 30% for KETRI 2455 and 3136, respectively (Table 4).
4. D i s c u s s i o n
There was a wide range of cloning success rates with the four T. evansi stocks examined. Barry et al. (1979) reported cloning success rates of 70% and 50% in Trypanosoma b. rhodesiense and Trypanosoma b. brucei respectively. These workers reported different cloning success rates in different species as compared to the results in this study where the success rates of cloning differed between different stocks of the same species. A greater success rate was obtained with the suramin-sensitive stocks of T. eoansi ( K E T R I 2454 and 2476) compared with the resistant stocks (KETRI 2455 and
Table 4 Subcloningsuccess rates of four T. evansi stocks, KETRI 2454, 2455, 2476 and 3136 KETRI no.
Resistance to suramin (mg kg- i )
No. of subclones established
No. of subcloning attempts
Subcloning success (%)
2454 Clone 1 2455 Clone 1 2476 Clone 1 3136 Clone 3
0.2 2 0.2 120
8 0 5 3
10 10 10 10
80 0 50 30
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3136). To a lesser extent, a similar trend was observed in sub-cloning, thus greater cloning success rates were observed when the clones of the suramin-sensitive stocks were sub-cloned. This success, however, was not directly related to the degree of suramin resistance, as the lowest success rate was obtained with KETRI 2455 and its clone which were resistant to suramin at dose rates of 10 and 2 mg k g - f body weight, respectively, and not with KETRI 3136 and its clone, resistant at dose rates of 160 and 120 mg kg-~ body weight respectively. Cloning success is determined by the ability of a single parasite to withstand the host's defence mechanisms and establish an infection. This may be related to some innate characteristics of the drug-sensitive trypanosome, such as a fast growth rate or high turnover of variable antigen types, which enables it to proliferate quickly in order to evade host-defence mechanisms. Although only four trypanosome stocks were investigated, the reduced cloning success rates obtained with the suramin-resistant trypanosomes suggests that they might establish less readily than sensitive stocks in vertebrate hosts. This agrees with the observation of Cantrell (1956) who studied the behaviour of drug-sensitive and resistant T. equiperdum parasites in mixed infections, and concluded that " m o s t drug resistant strains are less well adapted to life in the absence of the drug than their unmodified parents". Since clones from a single trypanosome stock showed a range of suramin sensitivities, field isolates are probably mixed populations as far as suramin sensitivity is concerned. This is the first time that T. evansi stocks have been shown to be heterogeneous populations with respect to drug sensitivity. Similar findings have been reported in other salivarian trypanosomes such as T. congolense (Peregrine et al., 1990). In that study, nine clones of the parent isolate had a spectrum of resistance to isometamidium chloride ranging from 1.5 to 5.1 mg kg-~ body weight while the range of resistance to diminazene aceturate was from 5.1 to 21 mg kg -1 . All of the clones of KETRI 2455 and 3136 and those investigated by Peregrine et al. (1990) had sensitivities to suramin and diminazene aceturate, respectively, which were lower than that of the parent isolate (35.7 mg kg-~). Similarly, the present observation with T. evansi KETRI 2454 and 2476, and the study on T. congolense using isometamidium (Peregrine et al., 1990), showed that some clones were more resistant than the parent stock. All other clones, however, had sensitivities which were lower than the parent stocks. Walker (1964) determined that ten resistant trypanosomes in a million are enough to give an isolate a resistant phenotype. One can thus assume that a heterogeneous stock must have at least this proportion of parasites, whose suramin sensitivity is the same as that of the parent isolate. If this assumption is correct, then there must have been at least this proportion of clones which gave identity to the stocks used in this experiment and, it is likely that they may have been among the ones inoculated into mice resulting in the failed cloning attempts. This would thus suggest that the resistant clones are less adapted to withstand the hostility of the host's defences than their sensitive counterparts as indicated by failure to establish infection. Hence, in the study of Peregrine et al. (1990), the clone which was more resistant to isometamidium chloride than the parent stock might thus have consisted of less than 0.001% of the total population. This argument would explain why most clones in this work and in the study of Peregrine et al. (1990) were of lower drug sensitivities compared to their parent clones.
M.W. Mutugi et aL / Veterinary Parasitology 60 (1995) 213-220
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In conclusion, this study has shown that four stocks of T. evansi consist of trypanosomes with sensitivities to suramin which are either the same as or lower than that of the parent stocks. Thus, with or without suramin, trypanosomes exhibited a range of suramin sensitivities and introduction of the drug would only select for the more resistant parasites. The success of cloning T. evansi in this study varied between stocks and clones of different suramin sensitivities and may have been related to the level of suramin sensitivity. The observation that drug-resistant trypanosomes were more difficult to clone and sub-clone compared to drug-sensitive ones suggests that a single resistant trypanosome is unable to survive in a mouse as readily as a sensitive one. Thus, cloning may be hindered by some inability of the drug-resistant parasites to establish infections as compared with drug-sensitive organisms. Although mice were used in this study, the findings observed may have implications in the establishment and maintenance of suramin resistance in T. evansi infecting camels under field conditions.
Acknowledgements This work was carried out as part of a Ph.D. programme at the University of Edinburgh by the first author funded by the Overseas Development Administration of the UK and Kenya Trypanosomiasis Research Institute (KETRI), and is published with the kind permission of the Director KETRI.
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