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Published in final edited form as: Acta Trop. 2015 Mar 16;146:95–100. doi: 10.1016/j.actatropica.2015.03.014

In vivo experimental drug resistance study in Trypanosoma vivax isolates from tsetse infested and non-tsetse infested areas of Northwest Ethiopia

Shimelis Dagnachew a,*, Getachew Terefe a, Getachew Abebe b, Dave Barry c, Richard McCulloch c, Bruno Goddeeris d
PMCID: PMC7612308  EMSID: EMS140938  PMID: 25792418

Abstract

Ethiopia, particularly in the Northwest region, is affected by both tsetse fly and non-tsetse fly transmitted trypanosomosis with a significant impact on livestock productivity. The control of trypanosomosis in Ethiopia 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 on the market for more than 40 years, this may have resulted in drug-resistance. Therefore, in vivo drug resistance tests on two Ethiopian isolates of Trypanosoma vivax were completed, one from an area where tsetse flies are present and one from an area where tsetse flies are not present. Twenty four cattle (Bos indicus) 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 of six 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 intravenously 3 × 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 treated at 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 of parasitaemia. Moreover, treatment did not improve diagnostic host markers of trypanosome infections in 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 trypanocidal drugs.

Keywords: Trypanosoma vivax, Drug resistance, Cattle, Northwest Ethiopia

1. Introduction

African animal trypanosomosis (AAT) is a debilitating and economically costly disease with a major impact on animal health in sub-Saharan Africa. Trypanosoma vivax, one of the trypanosome species responsible for most cattle disease (the other being Trypanosoma congolense), infects a wide host range including cattle, goats, horses and donkeys and is transmitted both cyclically by tsetse flies and mechanically by other biting flies (Hoare, 1972; Swallow, 2002; Mattioli et al., 2004). Ethiopia, particularly the Northwest region, is affected by both tsetse (cyclical) and non-tsetse (mechanical) transmitted trypanosomosis due to T. vivax with an associated impact on livestock productivity (Abebe and Jobre, 1996; Abebe, 2005; Sinshaw et al., 2006; Fikru et al., 2012). Trypanosomosis is controlled either by vector control or parasite control, or a combination of both. Parasite control currently relies on a small group of established trypanocidal compounds, and new compounds are unlikely to become available in the near future (Barrett et al., 2004). Consequently, the use of these drugs must be carefully monitored and trypanosome populations need to be screened regularly for the appearance of drug-resistant parasites. Several methods have been developed during recent years for the detection of drug resistance in trypanosomes. Most of the in vivo and in vitro assays used for this purpose are suitable for the determination of drug resistance of a small number of isolates, but are less appropriate for large-scale screening. However, there is increasing evidence that the effectiveness of chemotherapy is becoming reduced by the widespread development of trypanosome drug resistance (Van den Bossche et al., 2000; Delespaux et al., 2002). Drug resistance to isometamidium chloride (ISM) is more widespread than to diminazene aceturate (DA) (Geerts et al., 2001), but increasingly there are reports of resistance to both drugs (Geerts and Holmes, 1998; Mungube et al., 2012). Recent reports (Sow et al., 2012; Vitouley et al., 2012) confirmed ISM and DA resistance in T. vivax and T. congolense in West Africa. In Ethiopia the appearance of drug-resistant try-panosomes has been reported by several authors (Mulugeta et al., 1997; Afework et al., 2000; Ademe and Abebe, 2000; Tewelde et al., 2004; Shimelis et al., 2008; Moti et al., 2012). However, the reports on drug resistance are focused mainly on T. congolense and the importance of drug resistance as a problem for T. vivax is less well studied. Therefore, the work in this study was undertaken to test and compare the drug resistance of T. vivax isolates from tsetse infested and non-tsetse infested areas of Northwest Ethiopia.

2. Materials and methods

2.1. Study area

The experimental study was carried out in a fly-proof animal unit at the premises of the College of Veterinary Medicine and Agriculture of Addis Ababa University. T. vivax parasites used for this experiment were obtained from naturally infected cattle in two areas: Jabitehenan district of Birsheleko area (ETBS1 – Ethiopia Birsheleko isolate 1), located at 10° 42'N and 37° 16'E with an altitude of 1500 m above sea level (m.a.s.l.) and ~380 km Northwest of Addis Ababa; and in Bahir Dar Zuria district (ETBD1 – Ethiopia Bahir Dar isolate 1), located at 11°36’N and 37°23’E with an altitude of 1840 m.a.s.l. and ~590 km Northwest of Addis Ababa (Fig. 1) (see Section 2.5 for isolate details). These areas were selected as data is available confirming that tsetse flies are present (Birsheleko) by Dagnachew et al. (2005) or are not present (Bahir Dar) by Sinshaw et al. (2006). Birsheleko comprises a variety of different vegetation types including savannah, woodland, riverine, forest and cultivated land while in Bahir Dar the vegetation is composed of cultivated land, horticulture and grass land. In both areas there are permanent as well as seasonal rivers.

Fig. 1. Map of administrative regions of Ethiopia and administrative districts of Amhara region showing the sampling sites World Food Program Vulnerability Analysis and Mapping Unit, Ethiopia, July 1998.

Fig. 1

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2.2. Experimental animals

Twenty four indigenous Zebu (Bos indicus) cattle (8 male and 16 female) aged between 9 and 12 months were purchased from a try-panosome free area (Debre Berehan: Northcentral Ethiopia, located at 9°4’N and 39°32'E with an altitude of 2840 m.a.s.l. and ~130 km north of Addis Ababa. The animals were transferred to a fly-proof isolation unit at the College of Veterinary Medicine and Agriculture of Addis Ababa University, which is located at 9°6′N and 37°15′E, with an altitude of 1920 m.a.s.l. and is ~47 km east of Addis Ababa. Animals were ear-tagged, and examined for the presence of try-panosomes and other internal and external parasites using parasitological techniques (Murray et al., 1977; Soulsby, 1982). In order to avoid occurrence of pneumonia associated with transport stress and change of environment, all animals on arrival was treated with Oxytetracycline (20% w/v; Chongqing Fangtong Animal Pharmaceutical Co., Ltd., China). All animals were also treated with Albendazole (2500 mg bolus; Chengdu Qiankum Veterinary Pharmaceuticals Co. Ltd., China) and Ivermectin (1% injection, Norbrook, UK) to control internal and external parasites. The animals were acclimatized to the new environment, handling and feeding conditions for one month prior to the beginning of the experiment. During the acclimatization period physiological parameters such as packed cell volume (PCV) and rectal temperature were measured at weekly intervals to generate base line data for the experimental study.

2.3. Feeding and animal management

Animals were fed grass hay and supplemented with concentrates of wheat bran and green Elephant grass. Water and mineral lick were freely available. The handling of animals during the experiment was based on international guiding principles for biomedical research involving animals, as proposed by the Council for International Organizations of Medical Sciences (1985). The research was authorized by the Animal Research Ethics Review Committee of the College of Veterinary Medicine and Agriculture of the Addis Ababa University (Permit No. VM/ERC/003/07/015).

2.4. Experimental design

The efficacy of recommended curative doses of diminazene aceturate (DA) and isometamidium chloride (ISM) were tested against T. vivax isolates in experimentally infected cattle based on previous established protocols (Eisler et al., 2001). The experimental animals were randomly assigned into four groups of six animals per group: group TT-DA were infected with T. vivax from tsetse infested area and treated with DA; group TT-ISM were infected with T. vivax from tsetse infested area and treated with ISM; group NT-DA were infected with T. vivax from non-tsetse infested area and treated with DA; and group NT-ISM were infected with T. vivax from non-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 Dar isolate 1) were confirmed as pure T. vivax by a screening PCR (Masiga et al., 1992) and compared by targeting microsatellite markers using six different primers sets (Duffy et al., 2009), and found to be similar in four different primer sets. However, strain characterization was not performed to ask if each is a clone of a specific strain. Cryostabilates were prepared from these isolates and cryopreserved in liquid nitrogen and thereafter the stabilates were propagated on donor calves. The level of parasitaemia in the donor animals was estimated according to the “rapid-matching method” described by Herbert and Lumsden (1976) to be used for the experimental infections. Each experimental animal received 3 mL of infected blood from the donor animal at 106 trypanosomes mL–1 via the intravenous route.

2.6. Trypanocidal drugs

The trypanocidal drugs used in the experiment were diminazene aceturate (Diminasan™, Lot No. PS012910, Exp. 04-2016, ALFASAN, Kuipersweg 9, Woerden, Holland) and isometamidium chloride (Veridium™, Lot No. 198A1, Exp. 06-2015, CEVA SANTE ANIMALE, Libourne-France). DA was injected as a 7% solution at dose of 3.5 mg/kg body weight, and ISM was injected as a 1% solution at a dose of 0.5 mg/kg of body weight. Sterile distilled water was used to dissolve appropriate quantities of the drugs before they were administered to animals. The drugs were administered through the intramuscular route on the basis of accurate body weight measurements taken immediately before treatment.

2.7. Parasitological examination, PCV determination and drug resistance tests

Animals were clinically examined daily after experimental infection. Presence of parasites and determination of PCV values were undertaken based on previously established protocols (Murray et al., 1977), and assessment was carried out daily for the first 14 days post infection (dpi), and thereafter twice a week until the end of the experiment.

For the drug resistance tests, after all animals of a group became parasitaemic they were treated with the recommended doses (described above) of DA or ISM. From the treatment date cattle were monitored for parasitaemia by the buffy coat technique (Murray et al., 1977) twice a week for 100 days. When relapse (detection of trypanosomes after drug treatment) was detected the animal was treated with a second different drug. If no relapse was detected 100 days after the first and the second trypanocidal drug administration, the treatment was considered successful and the trypanosomes sensitive to drug treatment. Relapse infections detected within 100 days of administration of a trypanocidal drug were taken as indicative of resistance. If relapse occurred in more than 20% of the cattle tested, the strain was considered resistant to the dose of drug used (Eisler et al., 2001).

3. Results

3.1. Parasitaemia and PCV values

Twenty-four indigenous, trypanosome-free Zebu cattle were infected with T. vivax in four groups of six animals. Two groups were infected with T. vivax from a tsetse infested area and treated with DA or ISM; these were denoted TT-DA and TT-ISM, respectively. The two other groups were infected with T. vivax from a non-tsetse infested area and treated with DA or ISM; these were denoted NT-DA and NT-ISM, respectively. Parasite source areas in Ethiopia are shown in Fig. 1. An acute form of trypanosomosis was observed until treatment was applied in all groups of infected animals. Parasitaemia became patent 5 days post infection (dpi), with the first peak was observed on 7 dpi and the highest peak observed on 14 dpi, when treatment was given in the NT groups. For the TT groups parasitaemia became patent later, on 12 dpi, as did the peaks: the first peak was observed on 14 dpi and the highest peak was on 21 dpi, when treatment was given. The mean PCV values were significantly different (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 values were significantly different (p = 0.009, 95%CI = 9.90–14.39) between pre-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 PCV values remained significantly low after treatment for the NT groups (14 dpi) and for the TT groups (21 dpi), as shown in Fig. 2A,B, never returning to the pre-infection levels.

Fig. 2.

Fig. 2

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Mean PCV values (measured in 2 and 7 days interval before and after treatment, respectively) in young Zebu cattle experimentally infected with Trypanosoma vivax isolates from tsetse infested (TT) and non-tsetse infested (NT) areas followed by treatment either by DA or ISM. (A) Mean ± SE PCV values for NT-DA and NT-ISM groups. (B) Mean ± SE PCV values for TT-DA and TT-ISM groups during the study period.

3.2. Drug resistance test

Prior to treatment, peak parasitaemia was detected in all infected cattle. When the cattle were treated with DA or ISM, the parasitaemia was significantly reduced after 24 h. However, the assessment of drug resistance tests revealed the occurrence of resistant strains in cattle treated with the recommended doses of both drugs. A total of nine cattle, four from the NT groups (two ISM treated, and two DA treated) and five from the TT groups (three ISM treated, and two DA treated), showed detectable relapses of parasitaemia, which began 35 and 42 days post-treatment, respectively (Fig. 3A,B). The mean wave of parasitaemia in all six animals prior to treatment is shown, with the mean of the relapsing 2–3 cattle shown post treatment. For the NT groups, relapse of parasitaemia was detected in two animals 35 and 40 days post-treatment with DA and in another two animals 37 and 41 days post-treatment with ISM. In the TT groups two animals relapsed 42 and 45 days post-treatment with DA, and three animals 43, 45 and 56 days post-treatment with ISM. Consequently, more than 20% of the experimental animals in each treatment group demonstrated relapses of parasitaemia indicating the presence of resistant strains against the recommended doses of the trypanocidal drugs tested according to the definitions of Eisler et al. (2001).

Fig. 3.

Fig. 3

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Waves of parasitaemia (examined in 2 and 7 days interval before and after treatment, respectively) in young zebu cattle experimentally infected with Trypanosoma vivax isolates from tsetse infested (TT) and non-tsetse infested (NT) areas followed by treatment either by diminazene aceturate (DA) or isometamidium chloride (ISM). It shows 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 with NT-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 and 41 dpt. (B) From a total of six cattle infected with TT-treated by DA two showed relapse at 42 and 45 dpt, and from a total of six cattle infected with TT-treated by DA three showed relapse at 43, 45 and 56 dpt.

4. Discussion

4.1. Development of parasitaemia and associated PCV values

An acute form of trypanosomosis was observed in the experimentally infected cattle with both isolates of T. vivax, from tsetse-infested or non-infested areas of Northwest Ethiopia. An earlier onset of parasitaemia was detected in the NT infected cattle compared to the TT infected cattle. This finding may have occurred simply because the artificial intravenous experimental infection route we used correlates more closely with the mechanical transmission mode of NT T. vivax than with the tsetse fly transmission route to which TT trypanosomes are adapted (Conner and Mulka, 1986). Alternatively, the early onset of parasitaemia in the NT infected cattle could be due to a faster growth rate of the NT parasite. Irrespective, the differing rates of parasitaemia onset are comparable with work on Trypanosoma brucei (Turner et al., 1995) and Plasmodium chabaudi (Spence et al., 2013), where in both cases it has been demonstrated that when these parasites are syringe passaged in rodents there is an increase in parasitaemia and virulence. This has been attributed to a lack of a reset when going through the cyclical vector. In this study, although parasites appeared in the blood more quickly, there was no evidence for increased virulence in the NT group relative to the TT group. In all infected groups, an increase in parasitaemia coincided with a drop in PCV, which had also been reported in other studies (Murray and Dexter, 1988; Sekoni et al., 1990; Akinwale et al., 1999). Anaemia associated with trypanosome infections is multifactorial and the relative contribution of each mechanism will differ according to the host–parasite model, the phase of anaemia development and the severity of infection. Rapid PCV recovery after treatment with trypanocidal drugs (Holmes and Jennings, 1976) was not observed in the present work. The overall mean PCV remained below the physiological value of 33% (Aiello, 1998) even after treatment. This might be due to the presence of drug resistant populations of trypanosomes belowthe limits of our microscopical detection, though emergence of trypanosomes from drug inaccessible sites, nutritional imbalance and/or reduced response of the bone marrow due to exhaustion when the infection runs a chronic course cannot be ruled out (Murray and Dexter, 1988).

4.2. Drug resistance tests

In the present study relapses of parasitaemia were detected for both isolates and for both drugs tested. This would indicate a drug resistance status for these two isolates of T. vivax against both DA and ISM, contrary to the view expressed by Fairclough (1963), who suggested that it is difficult to induce resistance to isometamidium chloride even by repeated low dosages (0.25–0.5 mg/kg body weight). From animals treated with DA at 3.5 mg/kg and ISM at 0.5 mg/kg body weight, relapse was detected in 33% (4/12) and 41.66% (5/12), respectively. These levels of relapse are in agreement with reports by Leeflang et al. (1976) during their studies of the infectivity of Nigerian isolates of T. vivax in Zebu cattle, where infections reappeared after treatment with 3.5 mg/kg diminazene aceturate. Rottcher and Schillinger (1985) also claimed that, in the coast province of Kenya, T. vivax isolates that caused hemorrhagic disease were resistant to diminazene aceturate at 3.5 mg/kg. Rowlands et al. (1993) also reported similar findings on field investigations in Ghibe, reporting trypanosome populations resistant with the dosages of 3.5 mg/kg body weight and 0.5 mg/kg body weight for DA and ISM, respectively. Similar findings were reported on the occurrence of drug resistance in goats experimentally infected with T. vivax against the appropriate curative doses of 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 were not detected in goats treated with DA at a dose of 7 mg/kg body weight and ISM at dose of 0.5 mg/kg body weight. Conversely, in naive Boran (B. indicus) cattle T. vivax and Trypanosoma brucei mixed isolates exhibited low pathogenicity and were sensitive to diminazene aceturate at 3.5 mg/kg body weight and isometamidium chloride at 0.5 mg/kg body weight (Olila et al., 2002). In the same study in goats, four out of the eight goats infected with the same isolates expressed low levels of resistance to the same doses of ISM, whereas all eight infected goats were sensitive to diminazene aceturate at 3.5 mg/kg body weight. The present finding is also in agreement with field detection of resistance to DA (3.5 mg/kg) and ISM (1 mg/kg) against T. vivax in Burkina Faso (Sow et al., 2012; Vitouley et al., 2012). Finally, our findings are also in agreement with earlier studies from northern Ivory Coast in cattle on transit from Mali and Burkina Faso and with high prevalence of T. vivax infection (Kupper and Wolters, 1983), which showed resistance to doses of 0.5–1 mg/kg isometamidium chloride.

5. Conclusion

The current study showed that Zebu cattle experimentally infected with T. vivax from tsetse and non-tsetse infested areas of Northwest Ethiopia develop acute trypanosomosis. Treatment of infected cattle with the recommend doses of trypanocides showed incomplete parasite clearance, consistent with the occurrence of resistant strains and adding to growing evidence that such resistance may be a problem. Animals infected with a T. vivax isolated from non-tsetse area showed, however, shorter prepatent and relapse periods compared to animals infected with an isolate from tsetse infested area. Further field and molecular studies are needed with more isolates and at higher doses of trypanocides to monitor the drug resistance and to understand the mechanisms involved.

Acknowledgments

The author would like to thank Bahir Dar Regional Veterinary Laboratory for logistic support and enthusiastic encouragement during the field work. This work was supported by Ethio-Belgium VLIR Project (grant No. ZEIN2006PR324), GALVmed, Robert S. McNamara Fellowships Program (RSM) of the World Bank and the thematic research project ‘Animal Health Improvement’ of Addis Ababa University.

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