• Loading metrics

Isometamidium chloride and homidium chloride fail to cure mice infected with Ethiopian Trypanosoma evansi type A and B

  • Gebrekrustos Mekonnen,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliations College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia, College of Veterinary Medicine, Samara University, Afar, Ethiopia

  • Elmi Fahiye Mohammed,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliations College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia, IGAD Sheikh Technical Veterinary School (ISTVS), Nairobi, Kenya

  • Weldu Kidane,

    Roles Data curation, Methodology, Resources, Writing – review & editing

    Affiliation College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia

  • Awol Nesibu,

    Roles Conceptualization, Supervision, Writing – review & editing

    Affiliation School of Veterinary Medicine, Wollo University, Dessie, Ethiopia

  • Hagos Yohannes,

    Roles Conceptualization, Supervision, Writing – review & editing

    Affiliation College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia

  • Nick Van Reet,

    Roles Conceptualization, Formal analysis, Methodology, Writing – review & editing

    Affiliation Institute of Tropical Medicine, Nationalesstraat 155, 2000 Antwerp, Belgium

  • Philippe Büscher,

    Roles Conceptualization, Funding acquisition, Methodology, Resources, Writing – review & editing

    Affiliation Institute of Tropical Medicine, Nationalesstraat 155, 2000 Antwerp, Belgium

  • Hadush Birhanu

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing

    Affiliation College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia

Isometamidium chloride and homidium chloride fail to cure mice infected with Ethiopian Trypanosoma evansi type A and B

  • Gebrekrustos Mekonnen, 
  • Elmi Fahiye Mohammed, 
  • Weldu Kidane, 
  • Awol Nesibu, 
  • Hagos Yohannes, 
  • Nick Van Reet, 
  • Philippe Büscher, 
  • Hadush Birhanu



Trypanosoma evansi is mechanically transmitted by biting flies and affects camels, equines, and other domestic and wild animals in which it causes a disease called surra. At least two types of Trypanosoma evansi circulate in Ethiopia: type A, which is present in Africa, Latin America and Asia, and type B, which is prevalent in Eastern Africa. Currently, no information is available about the drug sensitivity of any Ethiopian T. evansi type.

Methodology/principal findings

This study was conducted with the objective of determining the in vivo drug sensitivity of two T. evansi type A and two type B stocks that were isolated from camels from the Tigray and Afar regions of Northern Ethiopia. We investigated the efficacy of four trypanocidal drugs to cure T. evansi infected mice: melarsamine hydrochloride (Cymelarsan), diminazene diaceturate (Veriben and Sequzene), isometamidium chloride (Veridium) and homidium chloride (Bovidium). Per experimental group, 6 mice were inoculated intraperitoneally with trypanosomes, treated at first peak parasitemia by daily drug injections for 4 consecutive days and followed-up for 60 days. Cymelarsan at 2 mg/kg and Veriben at 20 mg/kg cured all mice infected with any T. evansi stock, while Sequzene at 20 mg/kg caused relapses in all T. evansi stocks. In contrast, Veridium and Bovidium at 1 mg/kg failed to cure any T. evansi infection in mice.


We conclude that mice infected with Ethiopian T. evansi can be cured with Cymelarsan and Veriben regardless of T. evansi type. In contrast, Veridium and Bovidium are not efficacious to cure any T. evansi type. Although innate resistance to phenanthridines was previously described for T. evansi type A, this report is the first study to show that this phenomenom also occurs in T. evansi type B infections.

Author summary

Surra is a vector borne disease in camels, horses, water buffaloes, cattle and other domestic animals caused by Trypanosoma (T.) evansi. This protozoan parasite is transmitted by biting flies such as tabanids and stable flies and is endemic in many countries in Northern and Eastern Africa, Latin America and Asia. Surra is responsible for high economic losses due to mortality and morbidity of draught animals and leads to animal trade restrictions in endemic regions. Control of surra is mainly based on the treatment of sick animals presenting clinical symptoms. In Ethiopia two different types of T. evansi (A and B) have been described, yet no data existed about the drug sensitivity of any T. evansi type. In this study, we show for the first time that T. evansi type B is naturally in vivo resistant to the phenanthridine class of trypanocidal drugs, a phenonomen that was previously described for T. evansi type A. All Ethiopian T. evansi types are sensitive to melarsamine hydrochloride and diminazene diaceturate. Unfortunately, the most efficacious drugs are either not registered in Ethiopia or escape quality control of the active substance in commercial drug formulations. Furthermore, the inefficacious drugs remain accessible on the market despite their toxicity for animals.


African trypanosomoses (AT) are neglected parasitic diseases of humans and animals caused by various subgenera of pathogenic trypanosomes (Trypanozoon, Dutonella and Nannomonas). While human African trypanosomosis (HAT) has reached the point where elimination is being envisaged, animal African trypanosomosis (AAT) is still one of the major parasitic disease constraints to animal productivity in sub-Saharan Africa causing an estimated annual loss between 0.7 and 4.5 billion USD [14]. In Ethiopia, AAT has been described as a major impediment to livestock development and agricultural production, contributing negatively to development in general and to food self-reliance efforts of the country in particular. Both tsetse-transmitted (TTAT) and non tsetse-transmitted African trypanosomiasis (NTTAT) are endemic to the country. TTAT are due to Trypanosoma (T). congolense, T. vivax, and T. brucei brucei, whereas NTTAT are due to mechanically transmitted T. evansi and T. vivax, and the sexually transmitted T. equiperdum [512].

Surra is the number one protozoan disease of camels and is caused by T. evansi. Infected camels and equines may die within 3 months after onset of the disease. Moreover, cattle, water buffalo, pigs, goat and sheep infected with T. evansi suffer from immunosuppression, resulting in increased susceptibility to other diseases and vaccination failure against classical swine fever and Pasteurella multocida [1315]. The distribution of the disease mainly coincides with that of camels in the semi-desert areas of the country [5,7,16,17].

The control of surra relies mainly on the use of the trypanocidal drugs: the diamidine diminazene diaceturate, phenanthridines such as homidium salts (homidium chloride and homidium bromide) and isometamidium chloride, and the arsenical melarsamine hydrochloride [1822]. Isometamidium chloride is mainly used as a prophylactic drug and provides on average 3 months protection against trypanosome infection. Homidium salts have limited prophylactic properties and are mainly used as therapeutic agent [23]. Diminazene diaceturate and melarsamine hydrochloride are exclusively used as therapeutic agents [24].

Control of AT through chemotherapeutics is challenged by the emergence of drug resistance [25,26]. Resistance of T. congolense to isometamidium treatment has been reported in various areas of Ethiopia [11,12,27]. Similarly, Hagos and co-workers reported on the resistance of T. equiperdum against diminazene diaceturate [28]. Till present, there is no published evidence for drug resistance in Ethiopian T. evansi. However, isometamidium treatment failures in T. evansi infections have been documented in Sudan, China, the Phillipines and Venezuela [22,2933]. Ethiopian T. evansi stocks are composed of at least two types that are grouped into T. evansi type A and T. evansi type B based on the restriction enzyme profile of the kDNA minicircles [34,35]. T. evansi isolates with minicircle type A usually have the RoTat 1.2 variable surface glycoprotein (VSG) and are the most abundant in East and West Africa, Latin America and Asia [5,3539]. In contrast, T. evansi type B is less common and so far has only been isolated from camels in Chad, Kenya and Ethiopia [5,34,35,3941]. In a former study, we isolated T. evansi type A and type B stocks from camels in the Afar and Tigray regions in Northern Ethiopia [5,41]. The present study was undertaken to investigate the in vivo drug sensitivity profiles of some of these T. evansi stocks in mice with regard to diminazene diaceturate, isometamidium chloride, homidium chloride and melarsamine hydrochloride.

Materials and methods

Ethical considerations

Handling and use of experimental mice was approved by the College of Veterinary Medicine, Mekelle University (CVM-CRC/21/08), in line with the National Research Ethics Review Guideline of the Ethiopian Ministry of Science and Technology, Addis Ababa, 2014.

T. evansi stocks

For this study, we used two T. evansi type A (MCAM/ET/2013/004 and MCAM/ET/2013/009) and two T. evansi type B (MCAM/ET/2013/010 and MCAM/ET/2013/014) stocks, that we previously isolated from dromedary camel in Tigray and Afar, Northern Ethiopia [41]. All four stocks were typed as dyskinetoplastic trypanosomes based on absence of amplification of kDNA maxicircle targets. In addition, MCAM/ET/2013/009 is a natural akinetoplastic stock based on absence of kDNA minicircle amplification and loss of kinetoplast DAPI staining [41].

Expansion of trypanosome populations in mice

Trypanosome cryostabilates were thawed in a water bath at 37°C for 5 min, mixed with an equal volume of phosphate buffered saline glucose (PSG; 7.5 g/l Na2HPO4.2H2O, 0.34 g/l NaH2PO4.H2O, 2.12 g/l NaCl, 10 g/l D-glucose, pH 8) and checked for viability and motility of trypanosomes using microscopy. Swiss albino female mice of 6–8 weeks old and weighing between 25 and 30 g, obtained from the laboratory animal facility of the College of Veterinary Medicine of Mekelle University, were inoculated intraperitoneally (IP) with 0.2 ml of the trypanosome suspension. The parasitemia was monitored following the Matching Method, i.e. 5 μl of blood was transferred onto a microscope slide, covered with a 24x24 mm cover slip, examined at 40x10 magnification and the number of parasites per field of view were estimated and converted to parasites per ml of blood [42]. At peak parasitaemia, the mice were anaesthetised and exsanguinated by heart puncture with a heparinised syringe. Blood was diluted in PSG to a concentration of 2 trypanosomes per field (about 8x107 trypanosomes/ml) prior to use for in vivo drug sensitivity testing.

In vivo drug sensitivity testing

Per experimental group, 6 mice were inoculated intraperitoneally (IP) with 2.5x107 living trypanosomes in PSG. Infection of each animal was confirmed individually by microscopy one day before treatment. Treatment started at day 4 post-infection and consisted of daily IP injections for 4 consecutive days with 0.1 ml/10g body weight (BW) 0.9% NaCl saline solution containing the appropriate concentration of drug. Five trypanocidal drugs were tested in this study. Melarsamine hydrochloride (MelCy; Cymelarsan), isometamidium chloride hydrochloride (ISM; Veridium), and diminazene diaceturate (DIM; Veriben) were procured in Europe. Diminazene diaceturate plus phenazone (DIM-SEQ; Sequzene) and homidium chloride (HOM; Bovidium) were procured from the local market in Shire Endaselasse, Western zone of Tigray regional state. All drugs, except MelCy, were assessed by the Animal Products, Veterinary Drug and Feed Quality Assessment Center in Addis Ababa (Ethiopia) for adherence to the physiciochemical characteristics stated by their manufacturers. The scientific name, trade name, origin and dosage of the drugs are presented in Table 1.

Table 1. Scientific name, trade name, provider and dosage of the trypanocidal drugs used in this study.

We tested the following doses: 0.125 mg/kg BW and 2 mg/kg BW MelCy, 1 mg/kg BW ISM, 20 mg/kg BW DIM or DIM-SEQ and 1 mg/kg BW HOM [28,43,46]. The control group consisted of infected mice that received 0.2 ml of saline solution [43].

Two days after the last treatment and subsequently once a week until day 60 post- treatment, each mouse was examined with the Matching Method for the presence of parasites. To detect subpatent parasitaemia, survivor mice were immunosuppressed with cyclophosphamide at 200 mg/kg BW (Endoxan, Baxter, Lessines, Belgium) 25 days post-treatment [47]. Relapsing mice were euthanised. At day 60 post-treatment, all mice that remained negative in microscopy, were tested by the microhaematocrit centrifugation technique (mHCT, 4 tubes per mouse) [48]. If negative in mHCT, all surviving mice were euthanised and their blood was collected on heparin by heart puncture. The blood of all mice from each group was pooled and run over a mini Anion Exchange Centrifugation Technique (mAECT) column to detect subpatent parasitaemia [49]. If negative in mAECT, the mice were considered to be cured.


Detailed data on the outcome of the mice after infection and treatment are given in S1 Table. All infected mice treated with 0.9% saline (controls) died between the onset of treatment and two days after treatment. Table 2 shows the observed number of relapses and the average day after treatment that relapses occurred. MelCy at 0.125 mg/kg BW cured only 2 out of 6 mice infected with MCAM/ET/2013/004 (type A) and none of the mice infected with MCAM/ET/2013/014 (type B). Therefore, this dose was not administered to the mice infected with the two other stocks. MelCy at a higher dose (2 mg/kg BW) and DIM at 20 mg/kg BW cured all mice infected with any T. evansi stock. Treatment with DIM-SEQ at 20 mg/kg BW caused relapses for all T. evansi stocks. HOM at 1 mg/kg BW failed to cure any mouse infected with any T. evansi stock, while ISM at 1 mg/kg BW cured 4 of the 6 mice infected with MCAM/ET/2013/10 and none of the mice infected with the other stocks. No particular difference was apparent in parasitemia during pretreatment and relapse, between the T. evansi type A and type B stocks.

Table 2. Drug sensitivity profile of T. evansi type A and T. evansi type B stocks against MelCy, DIM, DIM-SEQ, ISM and HOM in mice.


This experimental study was conducted with the objective to determine the in vivo drug sensitivity profile in a mouse model of some recently isolated T. evansi stocks from Ethiopia. We performed the single-dose test, using the recommended dosages of DIM and ISM to discriminate resistant from sensitive strains, as described by Eisler et al [43], yet we extended the treatment regimen from 1 to 4 consecutive daily administrations to increase drug availability. The experiment terminated after a 60 days follow-up period, including immunosuppression on day 25 after treatment to reveal cryptic ongoing infections that may otherwise remain undetectable [46,47]. This long follow-up is necessary since relapses may occur after one month. Unfortunately, some mice died before the end of treatment, demonstrating the high virulence of some T. evansi stocks and the inefficacy of the drug used. For further studies with these T. evansi stocks, lower infection doses or earlier start of treatment should be considered. Also, for follow-up we recommend to use more sensitive tests than the Matching Method, such the mHCT, which was used only after 60 days of follow-up, or even PCR. As we performed a single-dose test to measure resistance or sensitivity only once for each drug and T. evansi strain, future in vivo drug sensitivity tests should apply a multi-dose test to more accurately define the level of resistance of each isolate.

Nevertheless, this is the first study to describe the in vivo drug sensitivity of Ethiopian T. evansi stocks. Specifically, in our study we tested two stocks of the common T. evansi type A, and two stocks of the elusive T. evansi type B, for which currently limited data are available on diagnosis, host range, clinical progress and treatment options.

In this study, we did not find evidence for arsenical or diamidine resistance in Ethiopian T. evansi. Both 2 mg/kg Cymelarsan and 20 mg/kg Veriben were able to cure all mice, infected with any T. evansi strain or subtype. However, more than half of the T. evansi infected mice, that were treated with Sequzene relapsed in the 4th week post- treatment, i.e. within maximum 8 days post-immunosuppression. Importantly, drug quality analysis of the used batches of both compounds reported that the purity of the compound complied with the manufacturer’s specifications (S1 Fig and S2 Fig). We have no conclusive answers to what caused this variability in cure rate. Given the fact that Veriben cured all T. evansi infected mice, it is unlikely that the difference with Sequzene can be attributed to diminazene resistance. Nevertheless, the Ethiopian stocks appear far less sensitive to cymelarsan and diminazene than reported for the Chinese isolate STIB 806K, where cure was obtained with < 0.125 mg/kg cymelarsan and with 2 mg/kg diminazene [46]. Previously, we showed that all Ethiopian T. evansi stocks used in this study appeared sensitive to cymelarsan and diminazene in in vitro drug testing [41]. Furthermore, all tested stocks carry a wild-type TevAT1 sequence, which encodes in T. evansi, T. equiperdum and in T. brucei for an aminopurine transporter (P2) known to import diminazene and MelCy [41,5054]. Arsenical, but not diminazene resistance, can also originate from mutations in the TbAQP2-AQP3 locus, by either deletion or chimerisation of TbAQP2 with TbAQP3, leading to reduced uptake of pentamidine and, to a lesser extent, of melarsen oxide [5557]. However, considering the susceptibility of the Ethiopian T. evansi to cymelarsan, this genetic locus was not further explored in this study.

Veridium (ISM) and Bovidium (HOM) at 1.0 mg/kg failed to cure completely any T. evansi infection in mice. Drug quality analysis of the used batches of Veridium and Bovidium indicated that the purity of the compounds complied with the manufacturer’s specifications (S3 Fig and S4 Fig). Both drugs belong to the phenanthridine class of trypanocidal agents and both are assumed to accumulate in the kinetoplast, inhibiting transcription and replication of kDNA [5860].

Recently, resistance to phenanthridines has been explained by genetic polymorphisms in the mitochondrial F1-ATPase y subunit, depletion of subunits of the vacuolar ATPase and absence of transport proteins that allow interaction between both ATPases [58,61,62]. Mutations in any of these proteins allow T. brucei to dispose of its kDNA, which in turn leads to phenanthridine and diamidine resistance [58,61,62]. Naturally dyskinetoplastic trypanosomes, such as T. evansi and T. equiperdum, have defined single nucleotide polymorphisms in the mitochondrial F1-ATPase y subunit that predispose them for complete loss of kDNA and thus cause innate resistance to primarily kDNA targeting drugs [6365]. Interestingly, therapeutic failure of ISM in mice and rats infected with T. evansi stocks from Indonesia and Nigeria was observed by others [66,67]. Furthermore, ISM treatment failures of T. evansi infections were previously reported in Sudan, China, the Philippines and Venezuela [30,68,69]. All Ethiopian T.evansi stocks in this study have corresponding polymorphisms in the F1-ATPase y subunit for type A and type B [41,64,65,70]. While the F1-ATPase y subunit A281del mutation, which characterises T. evansi subtype A, could be clearly linked to dyskinetoplasty and ISM resistance by genetic studies in T. brucei, similar studies could not confirm the effect of the F1-ATPase y subunit M282L mutation, which characterises T. evansi subtype B [62]. In this report, we provide for the first time evidence that T. evansi type B, like T. evansi type A, are naturally in vivo resistant to the phenanthridine class of trypanocidal drugs, despite earlier evidence that both types can be killed in vitro by ISM [41]. Interestingly, for T. evansi there appears to be no correlation between in vitro and in vivo ISM sensitivity. This phenomenon was already noted decades ago [21].


We conclude that Ethiopian T. evansi can be treated in mice by diminazene and MelCy regardless of T. evansi type and presence of kinetoplast. However, measures should be taken by the Ethiopian Veterinary Drug and Animal Feed Administration and Control Authority (VDAFACA) to create market access to Cymelarsan, which is currently not registered in Ethiopia, and to ensure consistent quality of commercial drug formulations that are available from the local markets. A recent study found that 27.3% of the diminaze diaceturate formulations and 29.4% of the isometamidium chloride formulations failed to comply with quality requirements as assessed in HPLC [71]. Furthermore, the phenanthridines isometamidium chloride and homidum salts are DNA intercalating agents that raise serious concerns of mutagenicity and are not well tolerated by camels [72]. Unfortunately, they are still in use for treating animals that provide beef and milk for human consumption [18,59,73].

Supporting information

S1 Table. Details on the outcome of mice infected with different T. evansi stocks and treated with different drugs.

ISM = isometamidium chloride hydrochloride, DIM = diminazene diaceturate, DIM-SEQ = diminazene diaceturate and phenazone granules, MelCy = melarsamine hydrochloride, HOM = homidium chloride. D = death, N = no parasites detected in blood, P = parasites detected in blood, T = treatment.


S1 Fig. Physicochemical test result of the used batch of Veriben.


S2 Fig. Physicochemical test result of the used batch of Sequzene.


S3 Fig. Physicochemical test result of the used batch of Veridium.


S4 Fig. Physicochemical test result of the used batch of Bovidium.



The authors would like to acknowledge Dr. Cyrille Chevtzoff (CEVA Santé Animale) for the generous gift of Veridium and Veriben used in this study. We acknowledge the staff of the Animal Products, Veterinary Drug and Feed Quality Assessment Centre in Addis Ababa for conducting the drug quality analysis.


  1. 1. Auty H, Torr SJ, Michoel T, Jayaraman S, Morrison LJ (2015) Cattle trypanosomosis: the diversity of trypanosomes and implications for disease epidemiology and control. Rev Sci Tech 34: 587–598. pmid:26601459
  2. 2. Morrison LJ, Vezza L, Rowan T, Hope JC (2016) Animal African Trypanosomiasis: Time to Increase Focus on Clinically Relevant Parasite and Host Species. Trends Parasitol. S1471-4922(16)30029-0 [pii];
  3. 3. Holmes P (2014) First WHO meeting of stakeholders on elimination of gambiense Human African Trypanosomiasis. PLoS Negl Trop Dis 8: e3244. PNTD-D-14-01158 [pii]. pmid:25340404
  4. 4. Shaw AP, Cecchi G, Wint GR, Mattioli RC, Robinson TP (2014) Mapping the economic benefits to livestock keepers from intervening against bovine trypanosomosis in Eastern Africa. Prev Vet Med 113: 197–210. S0167-5877(13)00334-6 [pii]; pmid:24275205
  5. 5. Birhanu H, Fikru R, Mussa S, Weldu K, Tadesse G, Ashenafi H et al (2015) Epidemiology of Trypanosoma evansi and Trypanosoma vivax in domestic animals from selected districts of Tigray and Afar regions, Northern Ethiopia. Parasit Vectors 8: 212. pmid:25889702
  6. 6. Fikru R, Goddeeris BM, Delespaux V, Moti Y, Tadesse A, Bekana M et al (2012) Widespread occurrence of Trypanosoma vivax in bovines of tsetse- as well as non-tsetse-infested regions of Ethiopia: a reason for concern? Vet Parasitol 190: 355–361. S0304-4017(12)00364-0 [pii]; pmid:22858227
  7. 7. Hagos A, Yilkal K, Esayass T, Alemu T, Fikru R, Fesseha G et al (2009) Parasitological and serological survey on trypanosomosis (surra) in camels in dry and wet areas of Bale Zone, Oromyia Region, Ethiopia. Rev Méd Vét 160: 569–573.
  8. 8. Hagos A, Degefa G, Yacob HT, Fikru R, Alemu T, Fesseha G et al (2010) Seroepidemiological survey of trypanozoon infection in horses in the suspected dourine-infected Bale highlands of the Oromia region, Ethiopia. Rev Sci Tech 29: 649–654. pmid:21309462
  9. 9. Hagos A, Abebe G, Büscher P, Goddeeris BM, Claes F (2010) Serological and parasitological survey of dourine in the Arsi-Bale highlands of Ethiopia. Trop Anim Health Prod 42: 769–776. pmid:19924557
  10. 10. Touratier L (2000) Challenges of non-tsetse transmitted animal trypanosomoses (NTTAT). An outline and some perspectives. Ann N Y Acad Sci 916: 237–239. pmid:11193626
  11. 11. Moti Y, Fikru R, Van den Abbeele J, Büscher P, Van den Bossche P, Duchateau L et al (2012) Ghibe river basin in Ethiopia: Present situation of trypanocidal drug resistance in Trypanosoma congolense using tests in mice and PCR-RFLP. Vet Parasitol. S0304-4017(12)00220-8 [pii]; pmid:22579499
  12. 12. Miruk A, Hagos A, Yacob HT, Asnake F, Basu AK (2008) Prevalence of bovine trypanosomosis and trypanocidal drug sensitivity studies on Trypanosoma congolense in Wolyta and Dawero zones of southern Ethiopia. Vet Parasitol 152: 141–147. S0304-4017(07)00661-9 [pii]; pmid:18207329
  13. 13. Holland WG, Do TT, Huong NT, Dung NT, Thanh NG, Vercruysse J et al (2003) The effect of Trypanosoma evansi infection on pig performance and vaccination against classical swine fever. Vet Parasitol 111: 115–123. pmid:12531288
  14. 14. Holland WG, My LN, Dung TV, Thanh NG, Tam PT, Vercruysse J et al (2001) The influence of T. evansi infection on the immuno-responsiveness of experimentally infected water buffaloes. Vet Parasitol 102: 225–234. pmid:11777602
  15. 15. Desquesnes M, Holzmüller P, Lai DH, Dargantes A, Lun ZR, Jittapalapong S (2013) Trypanosoma evansi and surra: a review and perspectives on origin, history, distribution, taxonomy, morphology, hosts, and pathogenic effects. Biomed Res Int 2013: ID 194176. pmid:24024184
  16. 16. Langridge W. P. (1976) A tsetse and trypanosomiasis survey of Ethiopia. London: Ministry of Overseas Development. 98 p.
  17. 17. Fikru R, Andualem Y, Getachew T, Menten J, Hasker E, Merga B et al (2015) Trypanosome infection in dromedary camels in Eastern Ethiopia: Prevalence, relative performance of diagnostic tools and host related risk factors. Vet Parasitol.
  18. 18. Holmes PH, Eisler MC, Geerts S (2004) Current chemotherapy of animal trypanosomiasis. In the Trypanosomiases. In: Maudlin I, Holmes PH, Miles MA, editors. Wallingford, UK: CABI International. pp. 431–444.
  19. 19. Geerts S. and Holmes P. H. (1998) Drug management and parasite resistance in bovine trypanosomiasis in Africa. Rome, Italy: FAO. 27 p.
  20. 20. Payne RC, Sukanto IP, Partoutomo S, Sitepu P, Jones TW (1994) Effect of suramin treatment on the productivity of feedlot cattle in a Trypanosoma evansi endemic area of Indonesia. Trop Anim Health Prod 26: 35–36. pmid:8009648
  21. 21. Zhang ZQ, Giroud C, Baltz T (1991) In vivo and in vitro sensitivity of Trypanosoma evansi and T. equiperdum to diminazene, suramin, MelCy, quinapyramine and isometamidium. Acta Trop 50: 101–110. pmid:1685865
  22. 22. Liao D, Shen J (2010) Studies of quinapyramine-resistance of Trypanosoma brucei evansi in China. Acta Trop 116: 173–177. S0001-706X(10)00227-5 [pii]; pmid:20813092
  23. 23. Peregrine AS, Gray MA, Moloo SK (1997) Cross-resistance associated with development of resistance to isometamidium in a clone of Trypanosoma congolense. Antimicrob Agents Chemother 41: 1604–1606. pmid:9210695
  24. 24. Berger BJ, Fairlamb AH (1994) Properties of melarsamine hydrochloride (Cymelarsan) in aqueous solution. Antimicrob Agents Chemother 38: 1298–1302. pmid:8092828
  25. 25. Geerts S, Holmes PH, Eisler MC, Diall O (2001) African bovine trypanosomiasis: the problem of drug resistance. Trends Parasitol 17: 25–28. S1471-4922(00)01827-4 [pii]. pmid:11137737
  26. 26. Delespaux V, Geysen D, Van den Bossche P, Geerts S (2008) Molecular tools for the rapid detection of drug resistance in animal trypanosomes. Trends Parasitol 24: 236–242. S1471-4922(08)00087-1 [pii]; pmid:18420457
  27. 27. Mulugeta W, Wilkes J, Mulatu W, Majiwa PA, Masake R, Peregrine AS (1997) Long-term occurrence of Trypanosoma congolense resistant to diminazene, isometamidium and homidium in cattle at Ghibe, Ethiopia. Acta Trop 64: 205–217. S0001-706X(96)00645-6 [pii]. pmid:9107367
  28. 28. Hagos A, Goddeeris BM, Yilkal K, Alemu T, Fikru R, Yacob HT et al (2010) Efficacy of Cymelarsan and Diminasan against Trypanosoma equiperdum infections in mice and horses. Vet Parasitol 171: 200–206. pmid:20417035
  29. 29. El Rayah IE, Kaminsky R, Schmid C, El Malik KH (1999) Drug resistance in Sudanese Trypanosoma evansi. Vet Parasitol 80: 281–287. pmid:9950334
  30. 30. Brun R, Lun ZR (1994) Drug sensitivity of Chinese Trypanosoma evansi and Trypanosoma equiperdum isolates. Vet Parasitol 52: 37–46. pmid:8030186
  31. 31. Zhou J, Shen J, Liao D, Zhou Y, Lin J (2004) Resistance to drug by different isolates Trypanosoma evansi in China. Acta Trop 90: 271–275. pmid:15099814
  32. 32. Zhang ZQ, Giroud C, Baltz T (1993) Trypanosoma evansi: in vivo and in vitro determination of trypanocide resistance profiles. Exp Parasitol 77: 387–394. S0014-4894(83)71098-2 [pii]; pmid:8253152
  33. 33. Kaminsky R, Schmid C, Lun ZR (1997) Susceptibility of dyskinetoplastic Trypanosoma evansi and T. equiperdum to isometamidium chloride. Parasitol Res 83: 816–818. pmid:9342750
  34. 34. Borst P, Fase-Fowler F, Gibson WC (1987) Kinetoplast DNA of Trypanosoma evansi. Mol Biochem Parasitol 23: 31–38. pmid:3033499
  35. 35. Njiru ZK, Constantine CC, Masiga DK, Reid SA, Thompson RC, Gibson WC (2006) Characterization of Trypanosoma evansi type B. Infect Genet Evol 6: 292–300. pmid:16157514
  36. 36. Bajyana Songa E, Paindavoine P, Wittouck E, Viseshakul N, Muldermans S, Steinert M et al (1990) Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates. Mol Biochem Parasitol 43: 167–179. pmid:1982554
  37. 37. Ou YC, Giroud C, Baltz T (1991) Kinetoplast DNA analysis of four Trypanosoma evansi strains. Mol Biochem Parasitol 46: 97–102. pmid:1677160
  38. 38. Lun ZR, Brun R, Gibson W (1992) Kinetoplast DNA and molecular karyotypes of Trypanosoma evansi and Trypanosoma equiperdum from China. Mol Biochem Parasitol 50: 189–196. pmid:1311051
  39. 39. Kamidi CM, Saarman NP, Dion K, Mireji PO, Ouma C, Murilla G et al (2017) Multiple evolutionary origins of Trypanosoma evansi in Kenya. PLoS Negl Trop Dis 11: e0005895. PNTD-D-17-00503 [pii]. pmid:28880965
  40. 40. Ngaira JM, Olembo NK, Njagi EN, Ngeranwa JJ (2005) The detection of non-RoTat 1.2 Trypanosoma evansi. Exp Parasitol 110: 30–38. pmid:15804376
  41. 41. Birhanu H, Gebrehiwot T, Goddeeris BM, Van Reet N, Büscher P (2016) New Trypanosoma evansi type B isolates from Ethiopian dromedary camels. PLoS Negl Trop Dis 10: 1–22.
  42. 42. Herbert WJ, Lumsden WH (1976) Trypanosoma brucei: A rapid ''matching'' method for estimating the host's parasitemia. Exp Parasitol 40: 427–431. pmid:976425
  43. 43. Eisler MC, Brandt J, Bauer B, Clausen PH, Delespaux V, Holmes PH et al (2001) Standardised tests in mice and cattle for the detection of drug resistance in tsetse-transmitted trypanosomes of African domestic cattle. Vet Parasitol 97: 171–182. S0304-4017(01)00415-0 [pii]. pmid:11390069
  44. 44. Connor R.J. (1992) The diagnosis, treatment and prevention of animal trypanosomosis under field condition. In FAO Animal Production and Health Paper 100, available from In.
  45. 45. Delespaux V, De Koning HP (2007) Drugs and drug resistance in African trypanosomiasis. Drug Resist Updat 10: 30–50. S1368-7646(07)00021-0 [pii]; pmid:17409013
  46. 46. Gillingwater K, Kumar A, Anbazhagan M, Boykin DW, Tidwell RR, Brun R (2009) In vivo investigations of selected diamidine compounds against Trypanosoma evansi using a mouse model. Antimicrob Agents Chemother 53: 5074–5079. AAC.00422-09 [pii]; pmid:19786604
  47. 47. Pyana PP, Van Reet N, Mumba-Ngoyi D, Ngay LI, Karhemere Bin Shamamba S, Büscher P (2014) Melarsoprol sensitivity profile of Trypanosoma brucei gambiense isolates from cured and relapsed sleeping sickness patients from the Democratic Republic of the Congo. PLoS Negl Trop Dis 8: e3212. pmid:25275572
  48. 48. Woo PTK (1970) The haematocrit centrifuge technique for the diagnosis of African trypanosomiasis. Acta Trop 27: 384–386. pmid:4396363
  49. 49. Büscher P, Mumba-Ngoyi D, Kabore J, Lejon V, Robays J, Jamonneau V et al (2009) Improved models of mini Anion Exchange Centrifugation Technique (mAECT) and Modified Single Centrifugation (MSC) for sleeping sickness diagnosis and staging. PLoS Negl Trop Dis 3: e471. pmid:19936296
  50. 50. Barrett MP, Zhang ZQ, Denise H, Giroud C, Baltz T (1995) A diamidine-resistant Trypanosoma equiperdum clone contains a P2 purine transporter with reduced substrate affinity. Mol Biochem Parasitol 73: 223–229. pmid:8577330
  51. 51. Matovu E, Stewart ML, Geiser F, Brun R, Maser P, Wallace LJ et al (2003) Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei. Eukaryot Cell 2: 1003–1008. pmid:14555482
  52. 52. Witola WH, Inoue N, Ohashi K, Onuma M (2004) RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate. Exp Parasitol 107: 47–57. pmid:15208037
  53. 53. Gillingwater K, Büscher P, Brun R (2007) Establishment of a panel of reference Trypanosoma evansi and Trypanosoma equiperdum strains for drug screening. Vet Parasitol 148: 114–121. pmid:17624671
  54. 54. Bridges DJ, Gould MK, Nerima B, Mäser P, Burchmore RJ, De Koning HP (2007) Loss of the high-affinity pentamidine transporter is responsible for high levels of cross-resistance between arsenical and diamidine drugs in African trypanosomes. Mol Pharmacol 71: 1098–1108. mol.106.031351 [pii]; pmid:17234896
  55. 55. Baker N, Glover L, Munday JC, Aguinaga AD, Barrett MP, De Koning HP et al (2012) Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes. Proc Natl Acad Sci U S A 109: 10996–11001. pmid:22711816
  56. 56. Graf FE, Ludin P, Wenzler T, Kaiser M, Brun R, Pyana PP et al (2013) Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol. PLoS Negl Trop Dis 7: e2475. pmid:24130910
  57. 57. Munday JC, Eze AA, Baker N, Glover L, Clucas C, Aguinaga AD et al (2014) Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs. J Antimicrob Chemother 69: 651–663. pmid:24235095
  58. 58. Eze AA, Gould MK, Munday JC, Tagoe DN, Stelmanis V, Schnaufer A et al (2016) Reduced mitochondrial membrane potential is a late adaptation of Trypanosoma brucei brucei to Isometamidium preceded by mutations in the gamma subunit of the F1Fo-ATPase. PLoS Negl Trop Dis 10: e0004791. PNTD-D-16-00228 [pii]. pmid:27518185
  59. 59. Roy CA, Bakshi R, Wang J, Yildirir G, Liu B, Pappas-Brown V et al (2010) The killing of African trypanosomes by ethidium bromide. PLoS Pathog 6: e1001226. pmid:21187912
  60. 60. Giordani F, Morrison LJ, Rowan TG, De Koning HP, Barrett MP (2016) The animal trypanosomiases and their chemotherapy: a review. Parasitology 143: 1862–1889. S0031182016001268 [pii]; pmid:27719692
  61. 61. Baker N, Hamilton G, Wilkes JM, Hutchinson S, Barrett MP, Horn D (2015) Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes. Proc Natl Acad Sci U S A 112: 9112–9117. pmid:26150481
  62. 62. Gould MK, Schnaufer AC (2014) Independence from Kinetoplast DNA maintenance and expression is associated with multidrug resistance in Trypanosoma brucei in vitro. Antimicrob Agents Chemother 58: 2925–2928. pmid:24550326
  63. 63. Schnaufer AC, Clark-Walker GD, Steinberg AG, Stuart K (2005) The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J 24: 4029–4040. pmid:16270030
  64. 64. Lai DH, Hashimi H, Lun ZR, Ayala FJ, Lukes J (2008) Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc Natl Acad Sci U S A 105: 1999–2004. pmid:18245376
  65. 65. Dean S, Gould MK, Dewar CE, Schnaufer AC (2013) Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes. Proc Natl Acad Sci U S A 110: 14741–14746. pmid:23959897
  66. 66. Jatau ID, Lawal AI, Agbede RIS, Abdurahman EM (2010) Efficacies of diminazene diaceturate and isometamidium chloride in Trypanosoma evansi infected rats. SJVS 8: 1–8.
  67. 67. Subekti DT, Yuniarto I, Sulinawati , Susiani H, Amaliah F, Santosa B (2015) Trypanocidals effectivity against some isolates of Trypanosoma evansi propagated in mice. JITV 20: 275–284.
  68. 68. Toro M, Leon E, Lopez R, Pallota F, García JA, Ruiz A (1983) Effect of isometamidium on infections by Trypanosoma vivax and T. evansi in experimentally-infected animals. Vet Parasitol 13: 35–43. pmid:6414154
  69. 69. Macaraeg B., Lazaro JV, Abes NS, Mingala CN (2013) In-vivo assessment of the effects of trypanocidal drugs against Trypanosoma evansi isolates from Philippine water buffaloes (Bubalus bubalis). Veterinarski Arhiv 83: 381–392.
  70. 70. Carnes J, Anupama A, Balmer O, Jackson A, Lewis M, Brown R et al (2015) Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty. PLoS Negl Trop Dis 9: e3404. pmid:25568942
  71. 71. Tekle T, Terefe G, Cherenet T, Ashenafi H, Akoda KG, Teko-Agbo A et al (2018) Aberrant use and poor quality of trypanocides: a risk for drug resistance in south western Ethiopia. BMC Vet Res 14: 4. [pii]. pmid:29304792
  72. 72. Ali BH, Hassan T (1986) Some observations on the toxicosis of isometamidium chloride (samorin) in camels. Vet Hum Toxicol 28: 424–426. pmid:3776085
  73. 73. Sutcliffe OB, Skellern GG, Araya F, Cannavan A, Sasanya JJ, Dungu B et al (2014) Animal trypanosomosis: making quality control of trypanocidal drugs possible. Rev Sci Tech 33: 813–830. pmid:25812206