A Study on Trypanocidal Drug Resistance: A Risk on Animal Health and Production in Jawi District of North West Ethiopia

Research Article

Austin J Vet Sci & Anim Husb. 2022; 9(4): 1101.

A Study on Trypanocidal Drug Resistance: A Risk on Animal Health and Production in Jawi District of North West Ethiopia

Ashagrie T1*, Zewde D1, Rufael T1, Mekonnen G1, Kefyalew H2, Selamu A3, Yenew A4 and Kassahun A4

1Animal Health Institute, Ethiopia

2Bahirdar Regional Veterinary Laboratory, Bahirdar, Ethiopia

3Finote selam Tsetse and Trypanosomiasis Investigation and Control Center, Finote selam, Ethiopia

4Jawi District Animal Health and Development Office, Jawi, Ethiopia

*Corresponding author: Tigist Ashagrie, Animal Health Institute, Ethiopia

Received: August 05, 2022; Accepted: September 02, Published: September 09, 2022


The study was conducted from February to June, 2021, with the objective to assess the occurrence of trypanocidal drug resistance in naturally infected bos indicus breed of cattle in hot spot villages of Jawi district, North Western Ethiopia. An initial cross-sectional prevalence study was conducted in four villages with the aim of finding at least 10% trypanosome parasite prevalence for further longitudinal drug susceptibility trial. Accordingly, out of 464 cattle examined from all villages, 53 (11.4%) animals were trypanosome-positive using a Buffy coat test method. For the appropriateness of monitoring and follow up of animals subjected to drug susceptibility trial and on the basis of village’s adjacency, the areas were merged in two sites (i.e. Achari and Kurey peasant associations). A 28-day field protocol study was used to estimate proportion of resistance to a recommended dose of 0.5 mg/kg Body Weight (bw) for Isometamidium Chloride (ISM) and 3.5mg/kg bw for Diminazene Aceturate (DIM). In this study, 52 trypanosome positive cattle were ear-tagged and allocated into two treatment groups. Group I, which consists 27 animals was treated with 7% solution of 3.5mg/kg bw DIM and the second group having 25 animals were subjected to 1% solution of 0.5 mg/kg bw ISM with 25 trypanosome free animals as a control. Before the actual treatment begin all animals grouped under treatment and control groups were subjected to deltamethrin 1% pour-on at day 0 on the animals back midline. Animals under trial were monitored for the status of trypanosomes and Packed Cell Volume (PCV) levels on days 14 and 28 post treatment. A treatment failure rate of 40% (10/25) forcattle’s treated with ISM was observed on day 28; whereas, 48.1% (13/27) DA treated animals on day 14 post-treatments were failed to clear the parasites. The results of the study confirmed the presence of drug resistance to the maximum recommended doses of ISM and DIM in the study district. Of all ISM and DIM treatment failures T. congolense accounted for 77.8% (7/9), 53.8% (7/13)); T. vivax (0%, 20%) and T. teleri (100%, 100%) respectively. Drug resistance has indeed been a considerable threat in each village of the study sites. DIM treated trypanosomepositive cattle showed a significantly increased (p < 0.001) PCV% from 19.9 % at day 0 to 21.2 % at day 14 and eventually to 23.8% at day 28. Similarly, ISM treated animals considerably increased (p<0.001) PCV% from 21.5% at day 0 to 23.9% at day 14 and then to 25.0% at day 28. However, the PCV% value for the negative control has no statistical variation (p=0.662) among the various days under investigation. Rational use of trypanocidal drugs and control of co-infections to exploit self-cure against resistant trypanosome populations are recommended. Furthermore, further study using advanced molecular techniques to explore possible pathways on development of drug resistance could be necessary.

Keywords: Bovine; Buffy coat; Drug resistance; Ethiopia; Trypanosomosis


Form the prevalent diseases of animal parasitism trypanosomiasis has a major complication to the development of animal health sector. It can affect health, reduce growth rate and birth weight, cause reproductive problems and reduce the usefulness of carcasses. Parasitic diseases are well known as main causes of economic losses in livestock worldwide [1,2]. Economic loss from parasitic disease is due to mortality, morbidity, reduced milk production and indirect impact on crop production through the availability and cost of animals that provide traction power. Among the existing parasitic diseases in Africa, Trypanosomosis (AAT) or Nagana is a daunting major problem in cattle and other ruminants [3].

Different trypanocidal drugs are used to prevent and treat trypanosomosis. In Africa about 35 million doses of drugs are used in each year, about 50-70 million animals at risk from trypanosomosis [4]. Since most of trypanocidal drugs have been in use for more than half a century, they can cause the appearance of the drug resistant strain of trypanosomes [5]. Trypanocidal drug resistance has been reported from 21 African countries. In ten of the 21 African countries, the occurrence of drug resistance to Diminazine Aceturate (DA), Isometamidium Chloride (ISM) and Homodium Bromide (HOM) has been reported [6]. Various researchers across East Africa reported resistance to currently available trypanocidal drugs and the approaches to the mechanisms of drug resistance in trypanosomes have been extensively reviewed [7]. In Ethiopia, ISM and DA have been used for several years. Trypanocidal resistance particularly against T. congolense infection was reported from Ghibe valley as early as in 1989 [8,9]. It was found that 11 of the 12 isolates tested were resistant in cattle to recommended doses of isometamidium and homidium. Since then, several other reports have emerged substantiating the widespread occurrence of trypanocidal drug resistance in many parts of the country [10-14].

A longitudinal study was followed for a 28 day field protocol which was a ‘Block treatment’ approach [5], that have been proposed to enable identification of probable resistance in the field, where by cattle in a particular location are split into control and treatment groups and followed for the detection of parasitemia. The two groups, naturally infected by trypanosome, were tested for trypanosomes positivity using Buffy coat technique [15]. Based on the protocol by [16], trypanocidal drug resistance study map ping have been documented by several epidemiological studies. [17] From Kénédougou Province of Burkina Faso, [18] from Eastern Zambia, [11] from Ethiopia, [19] from Guinea and south-eastern Mali and [20] from Ghana are some examples. In this regard, use of trypanocidal drugs must be carefully monitored and trypanosome populations need to be screened regularly for the appearance of drug resistant parasites. Based on the above concepts, we used to conduct in vivo resistivity test based on [21] in a 4 - week longitudinal survey. This was effective in areas where trypanosomosis risk was high (prevalence is >10%) [22]. for that reason, trypanosome drug resistance study was conducted in Jawi district where tsetse transmitted trypanosomosis become a serious threat for livestock production and agricultural activity [23,24]. The objective of the study in this district was todetermine the prevalence and assess magnitude of trypanocidal susceptibility in naturally infected cattle of the district.

Materials and Method

Study Area

The current study was conducted on purposively selected four village of Jawi district in Awi zone. The selection criteria was based on the extent of the existing problems, the complaints of farmers and the level of medium to high tsetse challenge in the area from the report of the field veterinarian as well as based on our recent parasitological findings in the area. Jawilies within the geographical location of 360 - 370E and 100380 to 110 30`N. The district has an altitude range from 648 to 1200 m.a.s.l. It has a climate which can be described as tropical with winter dry season. The agro-ecological area of the district has a warm and humid lowland zone around the area of the Belles River. The mean annual temperature varies between 25-400C with mean annual rain fall of 1569 mm. The livestock population of the district comprises about 70,403 cattle, 6,549 sheep, 24,995 goats, 1,232 equines, 30,997 poultry and 7,520 bee hives [25].

Study Animal

The study animal was indigenous zebu cattle reared extensively on communal grazing lands which were the principal feed source for cattle and other livestock during rainy season and crop residue were the major supplement available after harvest time. Only cattle’s aged above 1 year was considered and selected for the trial and allowed to graze together at the fringes of crop fields and fallow lands. They shared the same watering point during day time and housed at night on their respective farms. Animals obtain water in the rainy season from seasonal rivers while in the dry season from perennial rivers flowing in their locality.

Study Design

Both cross sectional and longitudinal study were conducted from February to June, 2021. The cross sectional study used to determine the prevalence of bovine trypanosomosis using microscopic/ parasitological and hematological examination while longitudinal study used to assess the level of trypanocidal drug resistance.

Sample Size Determination

To determine the prevalence of the disease in the Village (district sample size) a [26] formula was applied with an expected prevalence of 50%, 5% absolute desired precision and95% confidence level. Accordingly, sample Size was calculated:

N = (1.96)² Pexp (1-Pexp)/d²

Where: N = required sample size

Pexp = expected prevalence

d = desired absolute precision

A total of 384 cattle should have been needed to determine the prevalence of the disease as well as the PCV value of parasitic and a parasitic animals. However, to increase the chance of getting much number of positive animals which were subjected for drug susceptibility trial a total of 464 cattle’s were examined using a simple random sampling technique, where; 126, 182, 84, 72 animals were sampled from each village of Kuray, Achare, Addisalem and G/Wuha. The four hot spot villages were purposively selected from Jawi district as the areas were located in an ideal habitat for tsetse fly breeding. Hence, 25 trypanosome free animals were selected as a negative control and of 53 positive animals screened using a Buffy coat technique, 27 was grouped as a treatment group for Diminazene Aceturate (DA) and 26 for Isometamidium Chloride (ISM) treatment group. The four study sites were categorized into two (Achari and Kurey) for the purpose of effective monitoring.

Drug Susceptibility Study

Of 53 positive animals screened using a Buffy coat technique (Woo, 1970), 27 was grouped as a treatment group for Diminazene Aceturate (DA), 26 for Isometamidium Chloride (ISM). Placebo animals were used to guide for the reliability and disclose for the occurrence of any bias along the study period. Immediately to the selection, all animals under investigation were ear tagged based on their group of study and subjected to a deltamethrin 1% (Smash) pour-on, with 1ml/10kg bw (according to the manufacturer’s recommendation) along the back para-midline of the animal from shoulder to the base of the tail using T-bar applicator. This is important to expel fly contacts which are responsible to cause a bias on the study as the trial was conducted on extensively reared cattle’s. Before the actual treatment begins the animals were re-tested at day 0 to exclude for the presence of any latent trypanosome parasites infected animal from negative control groups and to realize the consistency of the parasites in positive animal groups. Indeed, of the 53 positive animals one was died before the actual treatment begun from ISM treatment group and the group was left with 25 animals. All the required parameters were recorded before and after the treatment; sex (male or female), body condition score (cachectic, lean and good [27], age group (<2 years, 2–5 years, and >5 years), coat color (red, black, white, gray or spoty), PCV (anemic <24% and non- anemic>24%) and species of trypanosome detected in the buffy coat (T. Conglense, T. vivax or mix and T. telerie).

A 28 day field protocol was used to estimate resistance to 3.5 mg/ kg/body weight diminazine aceturate and to 0.5 mg/kg/body weight isometamidium chloride in trypanosome positive cattle as described by [21]. Animals were randomly allocated to Diminazene (DIM) and Isometamidium (ISM) treatment groups. The animal’s weights were estimate during a weighing band as described by [27] to determine the dose of the drugs to be administered. All animals in group DIM received 3.5 mg/kg body weight of 7% solution Diminazine aceturate (DIMINAT®, Hebei Huarun, China) whereas group ISM was treated with 0.5mg/kg body weight of 1% solution Isometamidium chloride (ISM) (TRYPASHISH®, Tarapur, India). The drugs were administered intramuscular in gluteal and trapezium muscle for DIM and ISM respectively by veterinarians wherethis day was considered as day 0. Treated cattle were monitored for trypanosomes and PCV on days 14 and 28 post-treatment using parasitological and hematological technique. However, DIM treatment response was considered only at day 14 post treatment due to its short curative activity [28] and any animals that remain positive was treated with double dose of 7mg/kg body weight and followed for the extra 14 days. At the end of experimental trial all positive animals was treated/cleared with double dose of DIM 7mg/kg body weight.

Parasitological Examination and PCV Determination

The micro haematocrite capillary tubes were filled with the blood sample and sealed at one end using cristaseal. The capillary tubes were centrifuged at 12,000 Revolutions Per Minute (rpm) for 5 minutes to concentrate the trypanosomes in the Buffy coat layer [22]. First, the Packed Cell Volume (PCV) was measured soon after centrifugation using the Hawksley microhaematocrit reader (Hawksley, Lancing, United Kingdom). Then the capillary tube was placed in a Woo viewing chamber with a cover slip of 24 mm×24 mm and the Buffycoat plasma junction was examined under the microscope for the presence of trypanosomes. Trypanosome species were identified based on their movement using x 40 objective lens and ×10 eye pieces. Animals with PCV less than 24% considered to be anemic [29].

Ethical Consideration

Consent obtained from cattle owners and the livestock Office of the Woreda. Cattles used as control group during efficacy trial were disclosed for the owner for its negativity from trypanosome parasite at the end of the study. All animals involved in this study were handled according to standard guidelines for the use and handling of animals in research.

Data Management and Analysis

All data collected from parasitological and hematological investigation, questionnaire survey and trypanocidal drug resistance trial result was entered into Microsoft excel software making the data ready for further advanced software analysis (STATA, version 13 Stata Corp College Station, Texas, USA). Descriptive statistical analysis was done to assess the parasitological and hematological findings. An independent t-test and paired t-test was used to compare the parasite clearance ability of the drugs and the mean PCV value of animals under trial. A chi-square was also used to determine the association of covariates with response variables for the prevalence study. A p-value of <0.05 was considered as statistically significant across the study.


Total Prevalence of Bovine Trypanosomosis in the Study Area

Of the 464 cattle that were examined in the 4 villages, 53 (11.4%) were trypanosome-positive (Table 1). The distribution of the disease across the villages were nearly similar with no significant difference (p=0.556). Among trypanosome-positive animals, 15% (8/53) were mixed infection for T. congelense and T. vivax and 7.5% (4/53) were T. telerie which is a large parasite in the field. Mean PCV in the studied cattle was 23.7% (95% CI: 23.3-24.1%) with almost no variation among the villages (Table 2). However, positivity for the parasite among anemic and non-anemic animals had a momentous variation (p=0.001) where anemic animals were more positive for the infection (41/258, 15.9%) than non-anemic animals (12/206, 5.8%) (Table 3). Among the trypanosome-positive cattle, those infected with T. congolense had relatively lower mean PCV value (18.5%) compared to the cattle infected with T.vivax (20.2%), mixed infection (18.8%) and T.telerie (21.5%) as showed in (Table 1).