Trypanosoma simiae: In vitro studies on drug susceptibility

301

Acta Tropica, 54(t993)30t--308

© 1993 Elsevier Science Publishers B.V. All rights reserved 0001-706X/93/$06.00 ACTROP 00325

Trypanosoma simiae: In vitro studies on drug susceptibility E. Zweygarth a, S.K.

Moloo b and

R.

Kaminsky

b

aKenya Trypanosomiasis Research Institute, KETRI, hTkuyu, Kenya, and bInternational Laboratoryfor Research on Animal Diseases, ILRA D, Nairobi, Kenya

Two Trypanosoma simiae stocks were initiated in culture with tsetse-derived metacyclics. They were propagated axenically as trypomastigote forms at 35°C in 4% CO2 in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomaI compounds. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. Diminazene aceturate, quinapyramine sulphate, DL-~-difluoromethylornithine,and Ro 15-0216 showed activity against the stocks. Suramin and Mel Cy showed little effect upon the growth of the parasite populations. Isometamidium chloride gave questionable results in the 24 h growth inhibition test, but the results of a tong-term viability assay indicated some degree of drug resistance (or drug tolerance). The results obtained herein correlate with observations obtained from in vivo studies in pigs. it is thus concluded that in many cases the cryptic nature of T. simiae rather than drug resistance is responsible for the failure of chemotherapy of simiaetrypanosomlasls m p~gs. Key words: Trypanosomasimiae; Trypomastigote; In vitro cultivation; Axenic cultivation; Drug susceptibility; Diminazene aceturate; Isometamidium chloride; Quinapyramine sulfate; Suramin; Mel Cy; DL-~-Difluoromethylornithine D FMO; Rot 5-0216

Introduction Trypanosoma simiae is the m o s t serious cause o f mortalities in domestic pigs in tsetse-infested Africa, The disease is characterized by an acute to hyperacute course. Since in m a n y cases drug treatment is often too late, prophylactic regimens are the m e t h o d o f choice, either by keeping the animals on high g r o u n d in tsetse-free areas, in fly-proof housing or by the use o f c h e m o p r o p h y l a c t i c drugs. Trypanosoma simiae has long been considered intractable by chemotherapeutic interventions (summarized by Stephen, 1966); this was mostly attributed to d r u g resistance (or drug tolerance) o f the parasites. However, it was shown that pigs remained uninfected when treated with diminazene aceturate or quinapyramine sulphate 6 h after parasite inoculation, whereas with either drug when treatment was delayed until the animals were parasitaemic relapses invariably occurred. This suggests that the parasites per se were drug-sensitive ( M a h a g a and R6ttcher, 1981), Also it was later demonstrated that the stock o f T, simiae used by M a h a g a and R6ttcher invades the central nervous system (CNS) o f the infected pigs. The inaccessiCorrespondence to." E, Zweygarth, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, Republic of South Africa.

302 bility of the drug to the parasite in this site was assumed to be the cause of the relapses frequently observed after treatment (Zweygarth and R6ttcher, 1987a). In vitro drug susceptibility tests may help to elucidate this aspect of chemotherapy of simiae-trypanosomiasis in pigs, that is whether drug resistance or the cryptic nature of the parasites causes the observed phenomena. In the present paper, the results of drug susceptibility tests are compared with (1) the results obtained by drug treatment of pigs infected with the same stocks, (2) the results obtained for T. b. brucei and T. b. evansi in vitro as well as in vivo in mice.

Materials and Methods

Trypanosome stock Trypanosoma simiae stocks CP 11 (EATRO 1875, K E T R I 2431) and CP 813 were isolated in Muhaka forest in Ukunda, Kenya. Stock CP 11 was isolated from Glossina austeni in September 1970, whereas stock CP 813 was isolated from Glossina pallidipes in 1981. Trypanosomes of both stocks were highly virulent in domestic pigs infected either by tsetse transmission or by syringe-passage. The pigs died within 3 6 days of the onset of parasitaemia. Infection of tsetse Two groups of 200 teneral male Glossina morsitans centralis from the I L R A D breeding colony were each allowed to feed once on a pig (Large White) infected with T. simiae CP 11 or CP 813 during the initial rise of parasitaemia. Thereafter the tsetse flies were maintained by feeding on rabbits. After 25 days, the tsetse were induced to probe on to warmed slides at 37 ° C. The salivary probes were examined under a phase contrast microscope to identify those with a mature infection. Trypanosome cultures Axenic cultures of bloodstream forms of both T. simiae stocks were initiated and propagated as described by Zweygarth et al. (1992). Briefly, bovine aortic endothelial cell monolayers at passage 25-28 were used as feeder cells. Cultures of bloodstream forms were initiated and propagated in a medium consisting of Eagle's minimum essential medium (MEM; Gibco, Paisley, UK) supplemented with 20% heatinactivated (56 ° C, 30 min) pig serum and 1% MEM non-essential amino acids (Gibco), containing 100 IU/ml penicillin, 100 tag/ml streptomycin, 0.2 mM adenosine, 0.1 mM 2-mercaptoethanol (Baltz et al., 1985) and 0.02 mM 2,9-dimethyl-4,7-diphenyl-l,10-phenanthroline disulfonate (bathocuproine sulfonate, BCS; Serva Feinbiochemica, Heidelberg, Germany) (Yabu et al., 1989) and 1 mM L-cysteine (Duszenko et al., 1985). Adaptation to axenic conditions was done by transferring 0.5 ml of trypanosome-containing supernatant from feeder layer cell cultures to culture wells containing 0.5 ml medium only. The cultures were maintained at 35°C in a humidified atmosphere of 4% CO2 in air.

303 Drugs

Diminazene aceturate (Berenil, Hoechst, Frankfurt, Germany), isometamidium chloride (Samorin, May & Baker, Dagenham, UK), quinapyramine sulphate (Trypacide, May & Baker), and suramin (Naganol, Bayer, Leverkusen, Germany) were purchased commercially. The following compounds were also tested: Mel Cy (Cymelarsan, Rh6ne M6rieux, Toulouse, France), Ro 15-0216, 2-(dimethylamino)-4'-[(1-methyl2-nitroimidazole-5-yl)methoxy] acetanilide (F. Hoffmann-La Roche, Basel, Switzerland), DL-~-difluoromethylornithine hydrochloride monohydrate (DFMO; Eflornithine; Ornidyl; Merrell Dow, Cincinnati, OH, USA). Growth inhibition test

The growth inhibition test was carried out as described by Kaminsky and Zweygarth (1989) for T. b. brucei and T. b. evansi with minor modifications. Suspensions of trypanosomes derived from axenic cultures were adjusted to a concentration of 2 x 105 ml and 375 gl were pipetted into a 48-well culture plate (Costar, Cambridge, MA, USA). An equal volume of 2 x concentrated drug solution in medium was added. Each drug concentration was tested in duplicate, and was repeated at least twice. After 24 h of incubation, aliquots were removed from each well and trypanosomes were counted in a Neubauer counting chamber. The number of generations in drug-treated cultures was calculated for each well and the relative growth of trypanosome populations was determined by comparison with the number of generations (100%) in control cultures. The drug concentration which inhibited growth of trypanosome populations by 50% (ICso) was determined using logit analysis, minimum chi-square method. Values > 100% and < 0% growth were rejected. Long-term in vitro viability test

The long-term viability test was performed as described previously for T. b. brucei (Kaminsky et al., 1989) with minor modifications. Suspensions of culture-derived trypanosomes were adjusted to a concentration of 2 x 10S/ml and 1 ml was pipetted into wells of a 24-well culture plate (Costar) and propagated in the presence of bovine aortic endothelial cells (BAE) as feeder layer cells. The wells were left overnight before the drug test was performed. Each concentration of isometamidium was tested in duplicate. Half of the medium was replaced daily by fresh, drugcontaining medium. Culture growth was evaluated for 10 consecutive days.

Results

The ICso values of the four commercial drugs and the three experimental compounds tested against T. simiae stock CP 11 and CP 813 in vitro are shown in Table 1. Five of the compounds showed antitrypanosomal activity in vitro whereas suramin and Mel Cy hardly affected growth of the trypanosomes even at maximum drug levels of 100 gg/ml and 0.5 gg/ml, respectively. The effect of isometamidium on the growth of T. simiae bloodstream forms is shown in Table 2. In the presence of 0.3 ng/ml isometamidium, trypanosomes of both stocks grew in an identical manner to those

304 TABLE 1 Susceptibility of Trypanosoma simiae CP 11 and CP 813 to antitrypanosomal c o m p o u n d s Drugs

IC5o (ktg/ml)

Antitrypanosomal standard drugs Diminazene Isometamidium Quinapyramine sulphate Suramin

CP l l

CP 813

0.025 0.053 0.0015 > 100

0.039 0.160 0.0015 > 100

New drugs or experimental c o m p o u n d s Mel Cy >0.5 DFMO 9.6 Ro 15-0216 0.13

>0.5 13.6 0.109

TABLE 2 Long-term in vitro viability assay of Trypanosoma simiae CP 11 and CP 813 bloodstream forms in the presence of isometamidium + , normal growth and normal shape of trypanosomes; ( + ) , reduced growth and/or abnormal forms; - , no surviving trypanosomes. Isometamidium concentration (ng/ml)

Period of culture (days) 1

2

3

4

5

6

7

8

9

o.3

+

+

+

+

+

+

+

1

+

+

+

+

+

+

+

+ (+)

+ (+

3 10

+

+

+ +

+ (+)

+ (+)

(+) (+)

(+

+

+ (+)

(+)

+

-

-

CP 813 0.3

I0

c P 11

+

+

+

+

+

+

+

+

+

1

+

+

+

+

+

+

+

+

+

3 10

+ +

+ +

+ (+)

+ (+)

+ (+)

(+) .

(+)

(+

.

(+) .

+

(+) (+)

+ +

(+)

.

in the control wells. Stock CP 11 was affected by the presence of I ng/ml isometamidium in the medium whereas stock CP 813 was not. In the presence of 3 ng/ml isometamidium, bloodstream form cultures of both stocks showed a reduction in growth beginning on day 7 and 6, respectively, and trypanosomes did not survive in culture beyond day 7 and 5 when the isometamidium concentration was 10 ng/ml.

Discussion

In vitro experiments on drug susceptibilities of two T. simiae stocks were performed in order to correlate the results with those previously reported for one of the stocks (CP 11) in vivo (Mahaga and R6ttcher, 1981; Zweygarth and R6ttcher, 1987b;

305 Zweygarth and R6ttcher, unpublished results). Since T. simiae can now be propagated as the bloodstream form in vitro (Zweygarth et al., 1992), cell cultures may facilitate drug susceptibility, or drug screening, studies and at least in part, replace the in vivo screening test in pigs. Diminazene was found to be very effective against both T. simiae stocks in vitro. The ICso values were in a similar range to those found for a diminazene-sensitive T. b. brucei (Kaminsky et al., 1989). Pigs were protected from the disease with as little as 1.25 mg/kg diminazene (usual recommended dose in cattle, 3.5 mg/kg) injected prior to needle inoculation with stock CP 11 (Mahaga and R6ttcher, 1981). Injecting diminazene at various dosages in the animals already parasitaemic quickly cleared the parasites from the circulation. However, in all the animals the infection relapsed after a few days (Bauer, 1955; Steel, 1968; Mahaga and R6ttcher, 1981; Zweygarth and R6ttcher, 1987b). This discrepancy in the effectiveness of diminazene was elucidated when it was found that T. simiae CP 11 invaded the CNS of pigs where they were probably inaccessible to diminazene (Zweygarth and R6ttcher, 1987a). This would explain the failure of diminazene in advanced T. simiae infections. The same pattern of diminazene efficacy in pigs was also demonstrated for T. simiae stock CP 813 (unpublished results). Therefore, the low ICso values obtained from the drug susceptibility test further support the suggestion that T. simiae is susceptible, but it is inaccessible to diminazene and many other veterinary trypanocides in advanced cases. The time of invasion of the CNS may vary with different T. simiae stocks, hence the great differences in the success rate of the drug reported in the literature. Previous experiments comparing isometamidium treatment in cultures of a susceptible and a resistant T. b. brucei showed similar ICso values (Kaminsky and Zweygarth, 1989) which made a precise classification of susceptibility to the drug in the organisms difficult. In view of the narrow margin between susceptible and resistant organisms, an ICso value obtained with the 24 h growth inhibition test might not be suitable for differentiation of isometamidium susceptibilities. The average ICso values for isometamidium against T. simiae in the present experiments (0.053 lag/ml for stock CP 11 and 0.16 lag/ml for stock CP 813) were difficult to correlate with results obtained with stock CP 11 in pigs (Mahaga and R6ttcher, 1981). Hence, a long-term viability assay was applied, which appears more appropriate as isometamidium is used primarily for prophylaxis. Trypanosoma simiae stock CP 813 could be propagated in vitro equally well in the presence or absence of 1 ng/ml isometamidium; as much as 3 ng/ml was required to reduce the growth, whereas 10 ng/ml killed all trypanosomes in the cultures after 5 days of incubation. In contrast, T. simiae stock CP 11 was even more sensitive in vitro than stock CP 813. In comparison with in vivo data reported for stock CP 11 in pigs showed that 5 mg/kg isometamidium given 6 h prior to infection did not protect the animals from a subsequent infection (Mahaga and R6ttcher, 1981). It is therefore likely that stock CP 813 too would resist treatment with isometamidium at a dose level of 5 mg/kg body weight. Quinapyramine prosalt showed patterns of efficacy similar to diminazene in the treatment of pigs, where the success was dependent on the period between infection and trypanocide injection (Mahaga and R6ttcher, 1981). Both stocks were equally susceptible in vitro as expressed by their ICso value of0.0015 lag/ml. When quinapyramine sulphate was mixed with suramin, a barely soluble complex was formed which

306

was successfully used as a prophylactic against T. simiae infections (Watson and Williamson, 1958; Noble, 1958; Stephen and Gray, 1960), whereby suramin slows down the release of quinapyramine into the circulation but the latter is the actual trypanocidal component of the mixture. Suramin, which was also tested in the in vitro system, was hardly effective when used alone, ICso values being above 100 lag/ml for both stocks, compared to IC5o values of about 22 gg/ml for a suramin-resistant T. b. evansi stock (Kaminsky and Zweygarth, 1989). The inadequacy of suramin for the treatment of T. simiae infections was also demonstrated with stock CP 11 in pigs in which 20 mg/kg suramin failed to protect against infection when administered 6 h after trypanosome inoculation (Mahaga and R6ttcher, 1981). Mel Cy, cymelarsan, a melaminyl-substituted phenylarsonate earmarked for the use against surra (Raynaud et al., 1989), was also included in the tests although it was known that Mel Cy was ineffective at therapeutic drug doses against T. congolense (Dennig et al., 1989). The expectation that Mel Cy is not effective against T. simiae was confirmed in vitro, since the ICso values were more than 35-times higher than values found for a resistant, and 240-times higher than for a sensitive T. b. brucei stock (Zweygarth and Kaminsky, 1990). Among the new antitrypanosomal compounds, DFMO deserves special attention. DFMO was effective against early and late stages of the T. b. gambiense disease in man (Van Nieuwenhowe et al., 1985). When tested in vitro against both stocks of T. simiae, DFMO gave ICs0 values which were only about half of those obtained for a sensitive T. b. brucei clone and a T. b. brucei stock in a similar in vitro system (Zweygarth and Kaminsky, 1991). However, DFMO has the disadvantage that it has to be administered by infusion or orally. Oral application in pigs is difficult as the drug uptake cannot be controlled. Nevertheless, when 0.3% DFMO in the drinking water was given 1 day after inoculation (stock CP 11) for l0 consecutive days, two out of three pigs did not develop the disease, whereas all three untreated control animals became parasitaemic and succumbed to the disease (Schillinger, McCann, R6ttcher, Zweygarth, unpublished results). The experimental nitroimidazole compound Ro 15-0216 gave similar ICso values with T. simiae as with different sensitive T. b. brucei stocks (Zweygarth et al., 1990). This compound has proved very effective when combined with diminazene, especially in 'late stage' of simiae-trypanosomiasis (Zweygarth and R6ttcher, 1987b). So far, this treatment scheme is to our knowledge the only reliable and reproducible therapeutic intervention to cure parasitaemic animals, in contrast to quinapyraminesuraminate which suppresses the parasitaemia rather than completely eliminating the parasites. In conclusion, the in vitro tests confirmed that diminazene, quinapyramine, DFMO and the experimental nitroimidazole Ro 15-0216 are potentially suitable compounds for the treatment of 'early' T. simiae infections, at least with the stocks used in the present studies. The tests further showed that isometamidium at the recommended dose is not efficacious. The results of the in vitro experiments possibly also suggest that many previous studies of chemotherapy of trypanosomiasis in pigs should be re-interpreted, since we now know that T. simiae does invade the CNS. Consequently, inaccessibility of the organism by trypanocides within the CNS may have been wrongly interpreted as drug resistance.

307

Acknowledgments T h e p a p e r is p u b l i s h e d by p e r m i s s i o n o f the D i r e c t o r , K e n y a T r y p a n o s o m i a s i s R e s e a r c h I n s t i t u t e ( K E T R I ) . T h e a u t h o r s w o u l d like to t h a n k M r . J.P. M u l a t i , M r . C. S a b w a , M r . J. K a b a t a a n d M r . J. M u i a for t h e i r v a l u a b l e help.

References Baltz, T., Baltz, D., Giroud, C. and Crockett, J. (1985) Cultivation in a semi-defined medium of animalinfective forms of Trypanosoma brucei, T. equiperdum, T. evansi, 7". rhodesiense, and T. gambiense. EMBO J. 4, 1273-1277. Bauer, F. (1955) Trypanosomen- und Babesienerkrankungen in Afrika und ihre Behandlung mit dem neuen PrS~parat 'Berenil'. Z. Tropenmed. Parasit. 6, 129-140. Dennig, H.K., Jennings, F.W.,Leroy, J.P., Payne, R.C. and Raynaud, J.P. (1989) Cymelarsan R drug trials in mice. International Scientific Council for Trypanosomiasis Research and Control. Publication No. 115, 489 491. Duszenko, M., Ferguson, M.A.J., Lamont, G.S., Rifkin, M.R. and Cross, G.A.M. (1985) Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro. J. Exp. Med. 162, 1256-1263. Kaminsky, R. and Zweygarth, E. 0989) Feeder layer-free in vitro assay for screening antitrypanosomal compounds against Trypanosoma brucei brucei and T. b. evansi. Antimicrob. Agents Chemother. 33, 881-885. Kaminsky, R., Chuma, F. and Zweygarth, E. (1989) Trypanosoma brucei brucei: Expression of drug resistance in vitro. Exp. Parasitol. 69, 281-289. Mahaga, M.T. and R6ttcher, D. (1981) Chemotherapy of Trypanosoma simiae in pigs.International Scientific Council for Trypanosomiasis Research and Control. Publication No. 112, 284 288. Noble, N.M. (1958) Antrycide suramin complex as a prophylactic against trypanosomiasis in pigs a field trial in Sierra Leone. Bull. epiz. Dis. Afr. 6, 379-384. Raynaud, J.P., Sones, R.K. and Friedheim, E.A.H. (1989) A review of cymelarsan R -a new treatment proposed for animal trypanosomiasis due to T. evansi and other trypanosomes of the T. brucei group. International Scientific Council for Trypanosomiasis Research and Control. Publication No. 115, 334-338. Steel, E.D. (1968) Experiments in the chemotherapy and chemoprophylaxis of T. simiae infections in pigs, in Kenya. International Scientific Council for Trypanosomiasis Research and Control. Publication No. 102, 119-121. Stephen, L.E. (1966) Pig trypanosomiasis in Africa. Review Series No. 8 of the Commonwealth Bureau of Animal Health, pp. 33-37, Farnham Royal, UK. Stephen, L.E. and Gray, A.R. (1960) Suramin complexes. VI: The prophylactic activity of antrycidesuramin complex and antrycide chloride against Trypanosoma simiae in pigs. Ann. Trop. Med. Parasitol. 54, 493 507. Van Nieuwenhowe, S., Schechter, P.J., Declercq, J., Bone, G., Burke, J. and Sjoerdsma, A. (1985) Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL
308 Cymelarsan :~')against susceptible and drug-resistant Trypanosoma brucei brucei and T. b. evansi. Trop. Med. Parasitol. 41,208-212. Zweygarth, E., Kaminsky, R., Sayer, P.D. and Van Nieuwenhove, S. (1990) Synergistic activity of 5-substituted 2-nitroimidazoles (Ro 15-0216 and benznidazole) and DX.-~-difluoromethyl-ornithine on Tr)Tanosoma brucei brucei. Ann. Soc. belge M~d. Trop. 70, 269-279. Zweygarth, E. and Kaminsky, R. (1991) Evaluation of DL-~-difluoromethylornithine against susceptible and drug-resistant Trypanosoma brucei brucei. Acta Trop. 48, 223-232. Zweygarth, E., Moloo, S.K., Kaminsky, R. and Gray, M.A. (1992) Axenic in vitro cultivation of Trypanosoma simiae bloodstream form trypomastigotes. Acta Trop. 52, 79-81.