Two Trypanosoma vivax stocks were initiated in culture with tsetse or culture-derived metacyclics. They were propagated axenically as bloodstream trypomastigotes at 35°C in 4% C0₂ in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds, namely diminazene aceturate, quinapyramine sulphate, DL-α-difluoromethylornithine, isometamidium chloride, suramin and mel Cy. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. All six drugs tested showed little effect upon the growth of the parasite populations. These results indicate that a 24-h growth-inhibition test was not suitable for determining the drug susceptibility of T. vivax stocks in vitro. Neither did the results correlate with those obtained with susceptible or resistant stocks of T. b. brucei, T. b. evansi or T. simiae described in the literature, or with the results of these two T. vivax stocks tested in cattle.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Kaminsky, R.

Moloo, S.K.

Zweygarth, Erich

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Two Trypanosoma vivax stocks were initiated in culture with tsetse or culture-derived metacyclics. They were propagated axenically as bloodstream trypomastigotes at 35°C in 4% C0₂ in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds, namely diminazene aceturate, quinapyramine sulphate, DL-α-difluoromethylornithine, isometamidium chloride, suramin and mel Cy. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. All six drugs tested showed little effect upon the growth of the parasite populations. These results indicate that a 24-h growth-inhibition test was not suitable for determining the drug susceptibility of T. vivax stocks in vitro. Neither did the results correlate with those obtained with susceptible or resistant stocks of T. b. brucei, T. b. evansi or T. simiae described in the literature, or with the results of these two T. vivax stocks tested in cattle.mn201

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Onderstepoort Journal of Veterinary Research, 61:189-191 (1994) RESEARCH COMMUNICATION Evaluation of a short-term in vitro growth-inhibition test to determine susceptibility of Trypanosoma vivax stocks to various trypanocides E. ZWEYGARTH1 , R. KAMINSKY2 and S.K. MOL002 ABSTRACT ZWEYGARTH, E., KAMINSKY, R. & MOLOO, S.K. 1994. Evaluationofashort-termin vitro growth-inhibition test to determine susceptibility of Trypanosoma vivax stocks to various trypanocides. Onderstepoort Journal of Veterinary Research , 61:189-191 Two Trypanosoma vivax stocks were initiated in culture with tsetse or culture-derived metacyclics. They were propagated axenically as bloodstream trypomastigotes at 35 oc in 4 % C02 in air. Populations of trypanosomes were incubated with various concentrations of antitrypanosomal compounds, namely diminazene aceturate, quinapyramine sulphate, DL-a-difluoromethylornithine, isometamidium chloride, suramin and mel Cy. Growth was monitored after 24 h of incubation and the growth inhibition was calculated. All six drugs tested showed little effect upon the growth ofthe parasite populations. These results indicate that a 24-h growth-inhibition test was not suitable for determining the drug susceptibility ofT. vivax stocks in vitro. Neither did the results correlate with those obtained with susceptible or resistant stocks ofT. b. brucei, T. b. evansi or T. simiae described in the literature, or with the results of these two T. vivax stocks tested in cattle. A long-term in vitro viability assay was recently used to examine Trypanosoma vivax stocks for their sus-ceptibility to isometamidium chloride (Zweygarth , Kaminsky & Gray 1991 b). These experiments showed that isometamidium-sensitive and -resistant T. vivax stocks could be distinguished in vitro. In the present study, we used parasite populations derived from a ' Kenya Trypanosomiasis Research Institute, KETRI , Kikuyu, Kenya 2 International Laboratory for Research on Animal Diseases, ILRAD, Nairobi, Kenya Correspondence address: Dr Erich Zweygarth, Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, 0110 South Africa Received 24 March 1994- Editor continuous axenic culture system of T. vivax (Zwey-garth, Gray & Kaminsky 1991 a) , to determine their susceptibility to various antitrypanosomal drugs in a short-term (24 h) growth-inhibition test (Kaminsky & Zweygarth 1989). Trypanosoma vivax stock CP 2171 (originally desig-nated as 1V 5) was isolated from a Friesian cow in 1986 in Bamburi, Kenya, and stock CP 2331 (origi-nally designated as 1V 3) was isolated from a cow in 1986 in Kipini , Kenya. Both the T. vivax stocks have been characterized for their drug susceptibility in cat-tle by Schonefeld, Rottcher & Moloo (1987). The re-sults are summarized in Table 1. The T. vivax stocks were propagated in a combina-tion of lscove's modified Dulbecco's and RPM I 1640 medium (IMDM + RPMI 1640 XT powder medium; 189 Evaluation of a short-term in vitro growth inhibition test TABLE 1 Susceptibility of Trypanosoma vivax stocks CP 2171 and TABLE 2 Susceptibility of Trypanosoma vivax stocks CP 2171 2331 to antitrypanosomal drugs in cattle* and 2331 to antitrypanosomal drugs in vitro Drug Cured/treated cattle (dosage in mg/kg) CP2171 CP 2331 Diminazene (3,5) 11/12 3/3 lsometamidium (2) 0/3 0/3 Quinapyramine sulphate (4,4) 0/3 3/3 * Sch6nefeld et at. 1987 Serva Feinbiochemica, Heidelberg, Germany) sup-plemented with 20% (v/v) heat-inactivated (56 oc. 30 min) bovine serum, containing, in addition, 2 mM L-glutamine, 100 IU/mt penicillin, 100 !J.g/mt streptomy-cin, 0,2 mM adenosine, 0,1 mM 2-mercaptoethanol (2-ME)(Baltz, Baltz, Giraud, Crockett 1985), 1 mM L-cysteine and 0,02 mM 2,9-dimethyl-4,7 -diphenyl-1, 10-phenanthroline disulfonic acid (bathocuproine sulfonate, BCS; Serva Feinbiochemica) (Yabu, Takayanagi & Sato 1989). To initiate bloodstream-form cultures ofT. vivax stock CP 2171 , tsetse-fly-derived metacyclics were obtained from infected Glossina morsitans centra/is, by placing the infected hypopharynges into a culture well so that the metacyclics moved out into the medium. The cul-ture-derived metacyclics (Kaminsky, Chuma, Zwey-garth, Kitosi & Moloo 1991) of stock CP 2331 were used to initiate bloodstream-form cultures. Metacyclics were separated from epimastigotes by the method of Hirumi, Nelson, Hirumi, Moloo & Nantulya (1985) . Su-pernatants from insect-form cultures were incubated with 10% (v/v) bovine plasma at 27 oc for 1 h, which caused agglutination of the non-infective culture forms. Metacyclics were then separated by passage through a 5-flm pore filter. Propagation of bloodstream-form cultures of T. vivax has been described previously (Zweygarth et at. 1991 a). Diminazene aceturate (Berenil , Hoechst AG, Frank-furt, Germany), isometamidium chloride (Samarin, May & Baker, Dagenham, U.K.) , quinapyramine sulphate (Trypacide, May & Baker, Dagenham, U.K.) , and suramin (Naganol, Bayer AG, Leverkusen, Germany) were purchased commercially. The following com-pounds were kindly donated by the following drug companies: Mel Cy (Cymelarsan, Rhone Merieux, Tou-louse, France), and DL-a-difluoromethylornithine hy-drochloride monohydrate (DFMO; eflornithine; Ornidyl; Merrell Dow, Cincinnati , Ohio, USA). The growth-inhibition test was carried out as described by Kaminsky & Zweygarth (1989) forT. b. brucei and T. b. evansi. Suspensions of trypanosomes derived from axenic cultures were adjusted to a concentration of 2 x 1 05 mt, and 375 ~-t l were pipetted into each well of a 48-well culture plate (Costar, Cambridge, Mass. , USA) . An equal volume of 2x concentrated drug solu-190 IC50 (~-tg/ml) Drugs CP 2171 CP 2331 Diminazene aceturate 4,34 0,43 lsometamidium chloride 4,14 1,50 Quinapyramine sulfate 0,54 0,25 Suramin 64,10 77,00 Cymelarsan 0,40 0,47 DFMO > 100 > 100 tion in medium was added. Each drug concentration was tested in duplicate, and was repeated at least twice. After 24 h of incubation, 500 [..l. l aliquots were removed from each well , transferred into disposable cups (Sarstedt, Numbrecht, Germany) , fixed with 6 ~-tl formalin (37 %), diluted with lsoton (Coulter Electron-ics, Nairobi, Kenya) and counted in a Coulter Counter model ZM (70 flm aperture). The number of genera-tions in drug-treated cultures was calculated for each well and the relative growth of trypanosome popu-lations was determined by comparison with the num-ber of generations (1 00 %) in control cultures. The results of the drug-susceptibility tests carried out for both T. vivax stocks are summarized in Table 2. Animals infected with either stock were cured with the recommended dose of 3,5 mg/kg diminazene (Schone-feld et at. 1987). However, the IC50 values of 0,43 and 4,34 [..tg/mt, respectively, are out of the range for those found for other diminazene-susceptible trypanosome species tested with the same system. Trypanosoma b. brucei, T. b. evansi (Kaminsky & Zweygarth 1989) and T. simiae (Zweygarth, Moloo & Kaminsky 1993) had IC50 values approximately ten times lower than T. vivax stock CP 2331 . The antitrypanosomal action of diminazene is believed to be partially due to the inhi-bition of the kinetoplast DNA synthesis (Newton & LePage 1967). Diminazene also inhibited S-adenosyi-L-methionine (AdoMet) decarboxylase in T. b. brucei, an enzyme for the biosynthesis of polyamines, and its inhibition might contribute to the overall efficacy of diminazene as a trypanocide (Bitonti, Dumont & Mc-Cann 1986). The combined action of diminazene on both targets within the trypanosome would probably be more deleterious than the single action on either of the two targets. Therefore it is possible that the Ado-Met decarboxylase of T. vivax is inhibited to a lesser extent than that of T. b. brucei or T. simiae . This would explain the great discrepancy of the in vitro results between T. vivax and the other trypanosome species examined. In vivo, however, the action on DNA synthesis would be sufficient because of the support of the host 's immune system to clear the parasites from the circulation. The average IC50 values of isometamidium forT. vivax in the present experiments, i.e. 4,14 ~-tg/mt for stock CP 2171 and 1 ,5~-tg/mt for stock CP 2331, indicate a high level of drug resistance in vitro. However, com-paring previous data from a long-term viability assay obtained with stock CP 2331 (Zweygarth et at. 1991 b) , we found that they were almost identical with results obtained for a stock ofT. simiae (CP 813) (Zweygarth et at. 1993). Surprisingly, with the short-term growth-inhibition assay on the IC50 basis, the T. vivax stock was almost ten times more resistant than T. simiae. Similarly, for quinapyramine sulphate the IC~0 values were more than 160 times higher than those round for T. simiae stocks (Zweygarth et at. 1993) although one of the T. vivax stocks (CP 2331) was eliminated at the recommended dose of 4,4 mg/kg in cattle. In view of the IC50 values obtained for a suramin-re-sistant T. b. evansi stock (22 ,ug/mQ (Kaminsky & Zweygarth 1989), suramin was not expected to elimi-nate T. vivax infections with their even higher IC50 val-ues of 64,1 and 77 ~-tg/ml, respectively. Trypanosoma vivax has therefore been considered as non-suscep-tible to suramin treatment, which could also be con-cluded from the in vitro results. Mel Cy, a melaminyl-substituted phenylarsonate ear-marked for the use against surra (Raynaud, Sones & Friedheim 1989), was included in the tests although mel Cy was ineffective against a mouse-infective T. vivax stock (unpublished results) . The fact that mel Cy is not effective against T. vivax was confirmed in vitro since the IC values were more than 26 and 34 times higher than 5fhose values for a resistant T. b. brucei stock (Zweygarth & Kaminsky 1990). The new antitrypanosomal compound, DFMO, which was effective in man at all stages of the Gambian sleeping sickness (Van Nieuwenhowe, Schechter, Declercq, Bone, Burke & Sjoerdsma 1985), showed only a little activity against both T. vivax stocks as expressed by its IC50 values of more than 1 00 ~Lg/mt, the highest concentration used, as growth was inhib-ited by about 25% only (data not shown) . In conclusion, the present experiments indicate that a short-term growth-inhibition test has its limitations in determining drug susceptibilities of T. vivax stocks in vitro. Whether this was due to the culture-test system, the peculiarity of the parasites examined, or both, needs to be elucidated. None of the results correlated with those obtained with susceptible or resistant stocks ofT. b. brucei, T. b. evansi, and T. simiae in vitro, nor with those of the T. vivax stocks used in cattle. ACKNOWLEDGEMENTS The authors would like to thank Messrs J.P. Mulati, F. Chuma, C. Sabwa, J. Kabata and J. Muia for their valuable help. E. ZWEYGARTH, R. KAMINSKY & S.K. MOLOO REFERENCES BALTZ, T. , BALTZ, D., GIROUD, C. & CROCKETT, J. 1985. Cul-tivation in a semi-defined medium of animal-infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO Journal, 4:1273- 1277. BITONTI , A.J. , DUMONT, J.A. & MCCANN, P.P. 1986.Characteri-zation of Trypanosoma brucei brucei S-adenosyi-L-methionine decarboxylase and its inhibition by Berenil , pentamidine and methylglyoxal bis(guanylhydrazone). Biochemistry Journal, 237:685--689. HIRUMI, K. , NELSON, R.T ., HIRUMI, H. , MOLOO, SK & NANTU-L YA, V. 1985. Characterization of Trypanosoma vivax metacyclic trypomastigotes propagated in vitro. Proceedings, VI/Interna-tional Congress of Protozoology, Abstract No. 46, Nairobi, Kenya. KAMINSKY, R. & ZWEYGARTH, E. 1989. Feeder layer-free in vitro assay for screening antitrypanosomal compounds against Trypanosoma brucei brucei and T. b. evansi. Antimicrobial Agents and Chemotherapy, 33:881 - 885. KAMINSKY, R., CHUMA, F. , ZWEYGARTH, E. , KITOSI , D.S. & MOLOO, S.K. 1991. The effect of isometamidium chloride on insect forms of drug-sensitive and drug-resistant stocks of Trypanosoma vivax: Studies in vitro and in tsetse. Parasitology Research , 77:13-17. NEWTON, B.A. & LEPAGE, R.W.F. 1967. Preferential inhibition of extranuclear deoxyribonucleic acid synthesis by the trypanocide Berenil. Biochemistry Journal , 105. RAYNAUD, J .P., SONES, K.R. & FRIEDHEIM, E.A.H. 1989. A review of cymelarsan®-a new treatment proposed for animal trypanosomiasis due to T. evansi and other trypanosomes of the T. bruceigroup. International Scientific CouncilforTrypanosorn1aS1S Research and Control. Publication No. 115:334--338. SCHONEFELD, A., ROTTCHER, D. & MOLOO, S.K. 1987. The sensitivity to trypanocidal drugs ofT rypanosoma vivax isolated in Kenya and Somalia. Tropical Medicine and Parasitology, 38:177- 180. VAN NIEUWENHOW E, S., SCHECHTER, P.J., DECLERCQ, J. , BONE, G. , BURKE, J. & SJOERDSMA, A. 1985. Treatment of gambiense sleeping sickness in the Sudan with oral DFMO (DL-alpha-difluorornethyl-ornithine), an inhibitor of ornithine decarbo-xylase; first field trial. Transactions of the Royal Society of Tropi-cal Medicine and Hygiene, 79:692- 698. YABU, Y ., TAKAYANAGI, T . & SATO, S . 1989. Long-term culture and cloning system for Trypanosoma brucei gambiense blood-stream forms in semi-defined medium in vitro. Parasitology Re-search, 76:93- 97. ZWEYGARTH, E. & KAMINSKY, R. 1990. Evaluation of an arsenical compound (RM 110, mel Cy, Cyrnelarsan®) against susceptible and drug-resistant Trypanosoma brucei brucei and T. b. evansi. Tropical Medicine and Parasitology, 41 :208- 212. ZWEYGARTH, E., GRAY, M.A. & KAMINSKY, R. 1991a.Axenic in vitro cultivation of Trypanosoma vivax trypomastigote forrns. Tropical Medicine and Parasitology, 42:45- 48. ZWEYGARTH, E., KAMINSKY, R. & GRAY, M.A. 1991b. ln vitro assessment of isornetarnidium chloride susceptibil ity of Trypanosoma vivax bloodstream forms. Parasitology Research , 77:714--716. ZWEYGARTH, E., MOLOO, S .K. & KAM INSKY, R. 1993. Trypanosoma simiae: In vitro studies on drug susceptibility. Acta Tropica, 54:301 - 308. 191 - - ----- - - ------

Evaluation of a short-term in vitro growth-inhibition test to determine susceptibility of Trypanosoma vivax stocks to various trypanocides

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Evaluation of a short-term in vitro growth-inhibition test to determine susceptibility of Trypanosoma vivax stocks to various trypanocides

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