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Acta Tropica 146 (2015) 95–100

Contents lists available at ScienceDirect

Acta Tropica

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n vivo experimental drug resistance study in Trypanosoma vivaxsolates from tsetse infested and non-tsetse infested areas oforthwest Ethiopia

himelis Dagnachewa,∗, Getachew Terefea, Getachew Abebeb, Dave Barryc,ichard McCullochc, Bruno Goddeerisd

Addis Ababa University, CVMA, P.O. Box 34, Debre Zeit, EthiopiaFood and Agriculture Organization of the United Nations, FAO, Addis Ababa, EthiopiaUniversity of Glasgow, CMVLS, G12 8TA Glasgow, United KingdomFaculty of Bioscience Engineering, KU Leuven, B-3001 Heverlee, Belgium

r t i c l e i n f o

rticle history:eceived 7 October 2014eceived in revised form 6 March 2015ccepted 9 March 2015vailable online 16 March 2015

eywords:rypanosoma vivaxrug resistanceattleorthwest Ethiopia

a b s t r a c t

Ethiopia, particularly in the Northwest region, is affected by both tsetse fly and non-tsetse fly transmit-ted trypanosomosis with a significant impact on livestock productivity. The control of trypanosomosis inEthiopia relies on either curative or prophylactic treatment of animals with diminazene aceturate (DA)or isometamidium chloride (ISM), respectively. However, since these two trypanocides have been onthe market for more than 40 years, this may have resulted in drug-resistance. Therefore, in vivo drugresistance tests on two Ethiopian isolates of Trypanosoma vivax were completed, one from an area wheretsetse flies are present and one from an area where tsetse flies are not present. Twenty four cattle (Bosindicus) aged between 6 and 12 months, purchased from a trypanosome-free area (Debre Brehan: North-central Ethiopia) and confirmed to be trypanosome-negative, were randomly assigned into four groups ofsix animals, which were infected with T. vivax isolated from a tsetse-infested or non-tsetse infested area,and in each case treated with curative doses of DA or ISM. Each animal were inoculated intravenously3 × 106 trypanosomes from donor animals. Parasitaemia became patent earlier in infections with non-tsetse T. vivax (∼7 days post-infection) than tsetse (∼14 days post-infection). Both groups were treatedat the highest peak parasitaemia with DA or ISM and nine cattle, four with non-tsetse T. vivax (two ISM-

and two DA-treated) and five with tsetse T. vivax (three ISM- and two DA-treated) showed relapses ofparasitaemia. Moreover, treatment did not improve diagnostic host markers of trypanosome infectionsin these animals. In conclusion, in vivo drug tests indicated the presence of resistant parasites (>20%of treated animals in each group relapsed) against recommended doses of both available trypanocidaldrugs.

© 2015 Published by Elsevier B.V.

. Introduction

African animal trypanosomosis (AAT) is a debilitating and eco-omically costly disease with a major impact on animal health in

ub-Saharan Africa. Trypanosoma vivax, one of the trypanosomepecies responsible for most cattle disease (the other being Try-anosoma congolense), infects a wide host range including cattle,

∗ Corresponding author at: Addis Ababa University, College of Veterinary Medicinend Agriculture, P.O. Box 34, Debre Zeit, Ethiopia. Tel.: +251 921681563;ax: +251 114339933.

E-mail addresses: dagne2121@gmail.com, nigatusd2424@gmail.comS. Dagnachew).

ttp://dx.doi.org/10.1016/j.actatropica.2015.03.014001-706X/© 2015 Published by Elsevier B.V.

goats, horses and donkeys and is transmitted both cyclically bytsetse flies and mechanically by other biting flies (Hoare, 1972;Swallow, 2002; Mattioli et al., 2004). Ethiopia, particularly theNorthwest region, is affected by both tsetse (cyclical) and non-tsetse (mechanical) transmitted trypanosomosis due to T. vivaxwith an associated impact on livestock productivity (Abebe andJobre, 1996; Abebe, 2005; Sinshaw et al., 2006; Fikru et al., 2012).Trypanosomosis is controlled either by vector control or para-site control, or a combination of both. Parasite control currentlyrelies on a small group of established trypanocidal compounds,

and new compounds are unlikely to become available in thenear future (Barrett et al., 2004). Consequently, the use of thesedrugs must be carefully monitored and trypanosome populationsneed to be screened regularly for the appearance of drug-resistant

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arasites. Several methods have been developed during recentears for the detection of drug resistance in trypanosomes. Most ofhe in vivo and in vitro assays used for this purpose are suitable forhe determination of drug resistance of a small number of isolates,ut are less appropriate for large-scale screening. However, there

s increasing evidence that the effectiveness of chemotherapy isecoming reduced by the widespread development of trypanosomerug resistance (Van den Bossche et al., 2000; Delespaux et al.,002). Drug resistance to isometamidium chloride (ISM) is moreidespread than to diminazene aceturate (DA) (Geerts et al., 2001),

ut increasingly there are reports of resistance to both drugs (Geertsnd Holmes, 1998; Mungube et al., 2012). Recent reports (Sow et al.,012; Vitouley et al., 2012) confirmed ISM and DA resistance in. vivax and T. congolense in West Africa. In Ethiopia the appear-nce of drug-resistant trypanosomes has been reported by severaluthors (Mulugeta et al., 1997; Afework et al., 2000; Ademe andbebe, 2000; Tewelde et al., 2004; Shimelis et al., 2008; Moti et al.,012). However, the reports on drug resistance are focused mainlyn T. congolense and the importance of drug resistance as a prob-em for T. vivax is less well studied. Therefore, the work in thistudy was undertaken to test and compare the drug resistance of T.ivax isolates from tsetse infested and non-tsetse infested areas oforthwest Ethiopia.

. Materials and methods

.1. Study area

The experimental study was carried out in a fly-proof animalnit at the premises of the College of Veterinary Medicine andgriculture of Addis Ababa University. T. vivax parasites used for

his experiment were obtained from naturally infected cattle inwo areas: Jabitehenan district of Birsheleko area (ETBS1 – Ethiopiairsheleko isolate 1), located at 10◦42′N and 37◦16′E with an alti-ude of 1500 m above sea level (m.a.s.l.) and ∼380 km Northwestf Addis Ababa; and in Bahir Dar Zuria district (ETBD1 – Ethiopiaahir Dar isolate 1), located at 11◦36′N and 37◦23′E with an altitudef 1840 m.a.s.l. and ∼590 km Northwest of Addis Ababa (Fig. 1) (seeection 2.5 for isolate details). These areas were selected as datas available confirming that tsetse flies are present (Birsheleko) byagnachew et al. (2005) or are not present (Bahir Dar) by Sinshawt al. (2006). Birsheleko comprises a variety of different vegetationypes including savannah, woodland, riverine, forest and cultivatedand while in Bahir Dar the vegetation is composed of cultivatedand, horticulture and grass land. In both areas there are permanents well as seasonal rivers.

.2. Experimental animals

Twenty four indigenous Zebu (Bos indicus) cattle (8 male and6 female) aged between 9 and 12 months were purchased from

trypanosome free area (Debre Berehan: Northcentral Ethiopia,ocated at 9◦4′N and 39◦32′E with an altitude of 2840 m.a.s.l. and130 km north of Addis Ababa. The animals were transferred to ay-proof isolation unit at the College of Veterinary Medicine andgriculture of Addis Ababa University, which is located at 9◦6′N and7◦15′E, with an altitude of 1920 m.a.s.l. and is ∼47 km east of Addisbaba. Animals were ear-tagged, and examined for the presencef trypanosomes and other internal and external parasites usingarasitological techniques (Murray et al., 1977; Soulsby, 1982). Inrder to avoid occurrence of pneumonia associated with trans-

ort stress and change of environment, all animals on arrival wasreated with Oxytetracycline (20% w/v; Chongqing Fangtong Ani-

al Pharmaceutical Co., Ltd., China). All animals were also treatedith Albendazole (2500 mg bolus; Chengdu Qiankum Veterinary

ica 146 (2015) 95–100

Pharmaceuticals Co. Ltd., China) and Ivermectin (1% injection, Nor-brook, UK) to control internal and external parasites. The animalswere acclimatized to the new environment, handling and feedingconditions for one month prior to the beginning of the experiment.During the acclimatization period physiological parameters such aspacked cell volume (PCV) and rectal temperature were measuredat weekly intervals to generate base line data for the experimentalstudy.

2.3. Feeding and animal management

Animals were fed grass hay and supplemented with concen-trates of wheat bran and green Elephant grass. Water and minerallick were freely available. The handling of animals during theexperiment was based on international guiding principles forbiomedical research involving animals, as proposed by the Councilfor International Organizations of Medical Sciences (1985). Theresearch was authorized by the Animal Research Ethics ReviewCommittee of the College of Veterinary Medicine and Agricultureof the Addis Ababa University (Permit No. VM/ERC/003/07/015).

2.4. Experimental design

The efficacy of recommended curative doses of diminazene ace-turate (DA) and isometamidium chloride (ISM) were tested againstT. vivax isolates in experimentally infected cattle based on previ-ous established protocols (Eisler et al., 2001). The experimentalanimals were randomly assigned into four groups of six animalsper group: group TT-DA were infected with T. vivax from tsetseinfested area and treated with DA; group TT-ISM were infected withT. vivax from tsetse infested area and treated with ISM; group NT-DA were infected with T. vivax from non-tsetse infested area andtreated with DA; and group NT-ISM were infected with T. vivax fromnon-tsetse infested area and treated with ISM.

2.5. Trypanosome isolates and challenge

T. vivax isolates orginated from naturally infected cattle (ETBS1– Ethiopia Birsheleko isolate 1), and (ETBD1 – Ethiopia Bahir Darisolate 1) were confirmed as pure T. vivax by a screening PCR(Masiga et al., 1992) and compared by targeting microsatellitemarkers using six different primers sets (Duffy et al., 2009), andfound to be similar in four different primer sets. However, straincharacterization was not performed to ask if each is a clone of aspecific strain. Cryostabilates were prepared from these isolatesand cryopreserved in liquid nitrogen and thereafter the stabilateswere propagated on donor calves. The level of parasitaemia in thedonor animals was estimated according to the “rapid-matchingmethod” described by Herbert and Lumsden (1976) to be used forthe experimental infections. Each experimental animal received3 mL of infected blood from the donor animal at 106 trypanosomesmL−1 via the intravenous route.

2.6. Trypanocidal drugs

The trypanocidal drugs used in the experiment were dimi-nazene aceturate (DiminasanTM, Lot No. PS012910, Exp. 04-2016,ALFASAN, Kuipersweg 9, Woerden, Holland) and isometamidiumchloride (VeridiumTM, Lot No. 198A1, Exp. 06-2015, CEVA SANTEANIMALE, Libourne-France). DA was injected as a 7% solution atdose of 3.5 mg/kg body weight, and ISM was injected as a 1% solu-tion at a dose of 0.5 mg/kg of body weight. Sterile distilled water

was used to dissolve appropriate quantities of the drugs beforethey were administered to animals. The drugs were administeredthrough the intramuscular route on the basis of accurate bodyweight measurements taken immediately before treatment.

S. Dagnachew et al. / Acta Tropica 146 (2015) 95–100 97

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.7. Parasitological examination, PCV determination and drugesistance tests

Animals were clinically examined daily after experimentalnfection. Presence of parasites and determination of PCV val-es were undertaken based on previously established protocolsMurray et al., 1977), and assessment was carried out daily for therst 14 days post infection (dpi), and thereafter twice a week untilhe end of the experiment.

For the drug resistance tests, after all animals of a group becamearasitaemic they were treated with the recommended dosesdescribed above) of DA or ISM. From the treatment date cat-le were monitored for parasitaemia by the buffy coat techniqueMurray et al., 1977) twice a week for 100 days. When relapsedetection of trypanosomes after drug treatment) was detectedhe animal was treated with a second different drug. If no relapseas detected 100 days after the first and the second trypanocidalrug administration, the treatment was considered successful andhe trypanosomes sensitive to drug treatment. Relapse infectionsetected within 100 days of administration of a trypanocidal drugere taken as indicative of resistance. If relapse occurred in more

han 20% of the cattle tested, the strain was considered resistant tohe dose of drug used (Eisler et al., 2001).

. Results

.1. Parasitaemia and PCV values

Twenty-four indigenous, trypanosome-free Zebu cattle werenfected with T. vivax in four groups of six animals. Two groups

ere infected with T. vivax from a tsetse infested area and treated

tive districts of Amhara region showing the sampling sites

with DA or ISM; these were denoted TT-DA and TT-ISM, respec-tively. The two other groups were infected with T. vivax froma non-tsetse infested area and treated with DA or ISM; thesewere denoted NT-DA and NT-ISM, respectively. Parasite sourceareas in Ethiopia are shown in Fig. 1. An acute form of try-panosomosis was observed until treatment was applied in allgroups of infected animals. Parasitaemia became patent 5 dayspost infection (dpi), with the first peak was observed on 7 dpiand the highest peak observed on 14 dpi, when treatment wasgiven in the NT groups. For the TT groups parasitaemia becamepatent later, on 12 dpi, as did the peaks: the first peak wasobserved on 14 dpi and the highest peak was on 21 dpi, whentreatment was given. The mean PCV values were significantlydifferent (p = 0.041, 95%CI = 2.28–22.95) between pre-infection(33.56 ± 2.32 and 33.43 ± 3.47; NT-DA and NT-ISM, respectively)and post-infection (20.76 ± 2.98 and 23.05 ± 4.19; NT-DA and NT-ISM, respectively) in both NT groups. Similarly, the mean PCV valueswere significantly different (p = 0.009, 95%CI = 9.90–14.39) betweenpre-infection (34.56 ± 4.87 and 33.72 ± 2.14; TT-DA and TT-ISM,respectively) and post-infection (23.56 ± 3.15 and 20.91 ± 5.05; TT-DA and TT-ISM, respectively) in both TT groups. The mean PCVvalues remained significantly low after treatment for the NT groups(14 dpi) and for the TT groups (21 dpi), as shown in Fig. 2A,B, neverreturning to the pre-infection levels.

3.2. Drug resistance test

Prior to treatment, peak parasitaemia was detected in allinfected cattle. When the cattle were treated with DA or ISM,the parasitaemia was significantly reduced after 24 h. However,

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ig. 2. Mean PCV values (measured in 2 and 7 days interval before and after treatmsolates from tsetse infested (TT) and non-tsetse infested (NT) areas followed by tre

ean ± SE PCV values for TT-DA and TT-ISM groups during the study period.

he assessment of drug resistance tests revealed the occurrence ofesistant strains in cattle treated with the recommended doses ofoth drugs. A total of nine cattle, four from the NT groups (two ISMreated, and two DA treated) and five from the TT groups (threeSM treated, and two DA treated), showed detectable relapsesf parasitaemia, which began 35 and 42 days post-treatment,espectively (Fig. 3A,B). The mean wave of parasitaemia in allix animals prior to treatment is shown, with the mean of theelapsing 2–3 cattle shown post treatment. For the NT groups,elapse of parasitaemia was detected in two animals 35 and 40ays post-treatment with DA and in another two animals 37 and1 days post-treatment with ISM. In the TT groups two animalselapsed 42 and 45 days post-treatment with DA, and three ani-als 43, 45 and 56 days post-treatment with ISM. Consequently,ore than 20% of the experimental animals in each treatment

roup demonstrated relapses of parasitaemia indicating the pres-nce of resistant strains against the recommended doses of therypanocidal drugs tested according to the definitions of Eisler et al.2001).

. Discussion

.1. Development of parasitaemia and associated PCV values

An acute form of trypanosomosis was observed in the exper-mentally infected cattle with both isolates of T. vivax, from

setse-infested or non-infested areas of Northwest Ethiopia. Anarlier onset of parasitaemia was detected in the NT infected cat-le compared to the TT infected cattle. This finding may haveccurred simply because the artificial intravenous experimental

espectively) in young Zebu cattle experimentally infected with Trypanosoma vivaxt either by DA or ISM. (A) Mean ± SE PCV values for NT-DA and NT-ISM groups. (B)

infection route we used correlates more closely with the mechan-ical transmission mode of NT T. vivax than with the tsetse flytransmission route to which TT trypanosomes are adapted (Connerand Mulka, 1986). Alternatively, the early onset of parasitaemiain the NT infected cattle could be due to a faster growth rate ofthe NT parasite. Irrespective, the differing rates of parasitaemiaonset are comparable with work on Trypanosoma brucei (Turneret al., 1995) and Plasmodium chabaudi (Spence et al., 2013), wherein both cases it has been demonstrated that when these parasitesare syringe passaged in rodents there is an increase in parasitaemiaand virulence. This has been attributed to a lack of a reset whengoing through the cyclical vector. In this study, although parasitesappeared in the blood more quickly, there was no evidence forincreased virulence in the NT group relative to the TT group. In allinfected groups, an increase in parasitaemia coincided with a dropin PCV, which had also been reported in other studies (Murray andDexter, 1988; Sekoni et al., 1990; Akinwale et al., 1999). Anaemiaassociated with trypanosome infections is multifactorial and therelative contribution of each mechanism will differ according tothe host–parasite model, the phase of anaemia development andthe severity of infection. Rapid PCV recovery after treatment withtrypanocidal drugs (Holmes and Jennings, 1976) was not observedin the present work. The overall mean PCV remained below thephysiological value of 33% (Aiello, 1998) even after treatment. Thismight be due to the presence of drug resistant populations of try-panosomes below the limits of our microscopical detection, though

emergence of trypanosomes from drug inaccessible sites, nutri-tional imbalance and/or reduced response of the bone marrow dueto exhaustion when the infection runs a chronic course cannot beruled out (Murray and Dexter, 1988).

S. Dagnachew et al. / Acta Tropica 146 (2015) 95–100 99

Fig. 3. Waves of parasitaemia (examined in 2 and 7 days interval before and after treatment, respectively) in young zebu cattle experimentally infected with Trypanosomavivax isolates from tsetse infested (TT) and non-tsetse infested (NT) areas followed by treatment either by diminazene aceturate (DA) or isometamidium chloride (ISM). Itshows the mean of parasitaemia in all six animals prior to treatment, but the mean of only the relapsing 2–3 cattle post treatment. (A) From a total of six cattle infected withNT-treated by DA two showed relapse at 35 and 40 days post-treatment (dpt), and from a total of six cattle infected with NT- treated by ISM two showed relapse at 37 and4 apse as

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1 dpt. (B) From a total of six cattle infected with TT-treated by DA two showed relhowed relapse at 43, 45 and 56 dpt.

.2. Drug resistance tests

In the present study relapses of parasitaemia were detected foroth isolates and for both drugs tested. This would indicate a drugesistance status for these two isolates of T. vivax against both DAnd ISM, contrary to the view expressed by Fairclough (1963), whouggested that it is difficult to induce resistance to isometamid-um chloride even by repeated low dosages (0.25–0.5 mg/kg body

eight). From animals treated with DA at 3.5 mg/kg and ISM at.5 mg/kg body weight, relapse was detected in 33% (4/12) and1.66% (5/12), respectively. These levels of relapse are in agree-ent with reports by Leeflang et al. (1976) during their studies

f the infectivity of Nigerian isolates of T. vivax in Zebu cattle,here infections reappeared after treatment with 3.5 mg/kg dim-

nazene aceturate. Rottcher and Schillinger (1985) also claimedhat, in the coast province of Kenya, T. vivax isolates that causedemorrhagic disease were resistant to diminazene aceturate at.5 mg/kg. Rowlands et al. (1993) also reported similar findings oneld investigations in Ghibe, reporting trypanosome populationsesistant with the dosages of 3.5 mg/kg body weight and 0.5 mg/kgody weight for DA and ISM, respectively. Similar findings wereeported on the occurrence of drug resistance in goats experimen-ally infected with T. vivax against the appropriate curative doses

f DA (3.5 mg/kg body weight) and ISM (0.25 mg/kg body weight)Desalegn et al., 2010). However, in the same study relapses wereot detected in goats treated with DA at a dose of 7 mg/kg bodyeight and ISM at dose of 0.5 mg/kg body weight. Conversely, in

t 42 and 45 dpt, and from a total of six cattle infected with TT-treated by DA three

naive Boran (B. indicus) cattle T. vivax and Trypanosoma brucei mixedisolates exhibited low pathogenicity and were sensitive to dim-inazene aceturate at 3.5 mg/kg body weight and isometamidiumchloride at 0.5 mg/kg body weight (Olila et al., 2002). In the samestudy in goats, four out of the eight goats infected with the sameisolates expressed low levels of resistance to the same doses ofISM, whereas all eight infected goats were sensitive to diminazeneaceturate at 3.5 mg/kg body weight. The present finding is also inagreement with field detection of resistance to DA (3.5 mg/kg) andISM (1 mg/kg) against T. vivax in Burkina Faso (Sow et al., 2012;Vitouley et al., 2012). Finally, our findings are also in agreementwith earlier studies from northern Ivory Coast in cattle on transitfrom Mali and Burkina Faso and with high prevalence of T. vivaxinfection (Kupper and Wolters, 1983), which showed resistance todoses of 0.5–1 mg/kg isometamidium chloride.

5. Conclusion

The current study showed that Zebu cattle experimentallyinfected with T. vivax from tsetse and non-tsetse infested areas ofNorthwest Ethiopia develop acute trypanosomosis. Treatment ofinfected cattle with the recommend doses of trypanocides showedincomplete parasite clearance, consistent with the occurrence of

resistant strains and adding to growing evidence that such resis-tance may be a problem. Animals infected with a T. vivax isolatedfrom non-tsetse area showed, however, shorter prepatent andrelapse periods compared to animals infected with an isolate from

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setse infested area. Further field and molecular studies are neededith more isolates and at higher doses of trypanocides to monitor

he drug resistance and to understand the mechanisms involved.

cknowledgments

The author would like to thank Bahir Dar Regional Veterinaryaboratory for logistic support and enthusiastic encouragementuring the field work. This work was supported by Ethio-BelgiumLIR Project (grant No. ZEIN2006PR324), GALVmed, Robert S.cNamara Fellowships Program (RSM) of the World Bank and the

hematic research project ‘Animal Health Improvement’ of Addisbaba University.

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