Epidemiology and Detection of Multiple Drug-Resistant Trypanosoma congolense in Zebu Cattle in Gurage Zone, Southwest Ethiopia

Background Trypanosomosis in animal is a major threat for the development of livestock sector in many Africa countries as a result it contributes for the prevailing severe hunger and poverty in most sub-Saharan African countries. The aim of this study is to determine the prevalence and drug susceptibility pattern of Trypanosoma congolense in selected districts in Gurage zone, Ethiopia. Methods A cross-sectional study was carried out in 2000 selected indigenous cattle and eighty (n=80) cattle were selected out of 2000 to carry out drug susceptibility test using longitudinal study. Animals were grouped equally into two (n=40) treatment groups and treated with isometamidium chloride (ISMM) and diminazene aceturate (DIM) according to the manufacturer’s instruction. Sample was collected and the microhaematocrit centrifugation technique was used to determine PCV while trypanosomes were detected using dark-ground phase-microscopy.


Introduction
In sub-Saharan Africa (SSA), tsetse y-transmitted African animal trypanosomosis (AAT) is the cause for multibillions of dollars loss annually [1]. Hunger and poverty in SSA is exacerbated due to AAT where it represents a serious impediment to sustainable agricultural rural development [2]. For instance, ATA threaten the survival of draught animals as a result 80% of arable land is tilled by the hand of the farmers which is not e cient to obtain the required productivity to maintain food and nutrition security in the SSA.
In Ethiopia, trypanosomosis is a major bottleneck to the agricultural development in general and livestock production and productivity in particular. An estimated area of 220,000Km 2 is potentially infected by tsetse ies due to high infestation by tsetse ies to previously free areas, [3]. T. conglense and T. vivax are the most prevalent trypanosma species in the country. Various researchers revealed that the prevalence of trypanosomosis in tsetse infested areas range from 11.85-37% [4][5][6][7][8] However, in non-tsetse infested areas of Northwest Ethiopia the prevalence of trypanosomosis was in the range of 2-9% [9][10][11].
Trypanosmiasis is controlled using a combination of different approaches such as chemotherapy, vector control and typanotorolant animals. Chemotherapy is and will continue to be preferred in the control of trypanosomiasis [12]. Currently, a small group of chemo-prophylactic and chemo-therapeutic compounds are in use with new anti-trypanosomal compounds [13]. Despite the use of various drugs to treat trypanosmosis, the rapid development of resistance to the available drugs presents a serious threat that will potentially render them ineffective in trypanosomosis control [14]. Moreover, the bene ts of increased trypanocide treatment frequency diminish rapidly with increasing resistance [15]. The dependence of smallholder livestock farming on drugs against trypanosomsis attributed to aggravating the development drug resistance and spread of the trypanosome [16][17].
The existence of multiple drug resistance trypanosome species were reported from different corners African countries where Trypanosma is endemic [18]. At present, 21 African countries has been reported the existence of resistance against trypanocidal drug such as diminazene aceturate (DA), isometamedium (ISM) and homodium has been reported in trypanosome populations in ten African countries [19,20] Drug Incubation Glossian Infective Test (DIGIT) proved to be extremely sensitive in detecting drugresistant T. congolense populations, but requires availability of tsetse ies. Genetic markers can be used to characterize trypanocidal drug resistance [21,22]. The practical application of genetic markers is still limited in many sub-Saharan African countries (SSA) due to the lack of appropriately equipped laboratories and skilled personnel. Some researcher recommends longitudinal study is reliable though it takes long (up to 90 days) to generate results [23,24].
Isometamidium chloride and diminazene aceturate has been used for more than 40 years in Ethiopia to treat cattle against T.congolense and T.vivax infection [25]. There are some studies on trypanocidal resistance particularly against T.congolense infection as reported in the Ghibe valley and other parts of infested areas with trypanosmosis. However, still there are limited information on occurrence of drug resistant trypanosome across Ethiopia is not well known. Therefore, this study was undertaken to detect multidrug resistance T. Congolese against commonly used trpanocidal drugs (ISMM and DIM) in south west Ethiopia.

Study Area Description
The study was carried out in selected districts in Gurage zone, south-west Ethiopia which is located between7 76'and8 45'N latitude and 37 46'and38 71'E longitude, and at altitude of 1500-1850 meter above sea level (Fig 1). The annual temperature and rainfall ranges are 11-22 C and 800-1200 mm with peaks in July and August, respectively. The society based on mixed farming system which include both crops and livestock production. The livestock population in the district accounted 40.204 bovine, 4.217 equine and 7.624 caprine, 1.825 ovine and 35.680 poultry.

Study animals
The study was carried out in freely grazing indigenous zebu cattle of both sexes in the selected study areas. Cattle aged 12 months and above were sampled and examined in the study districts as recommended by [25]. Animals for the drug sensitivity analysis were known to be infected with T. congulense.

Study design
A cross-sectional study was carried out to determine the prevalence of T. congulense in the study areas. Accordingly, ve (Fig 1) out of 40 villages were selected depending on the prevalence of trypanosomes. Blood samples were collected from 2000 cattle in ve villages and screened for T. congulense using Woo test techniques in order to determine the prevalence of Trypanosome in the study village [26].

Study on Drug sensitivity
Longitudinal study was conducted from November-December 2013 on 80 known T. congulense positive cattle which were randomly allocated into two equal treatment groups. An abbreviated 28-day eld protocol was used to estimate resistance to 3.5 mg/kg and 7.0 mg/kg bw DIM and to 0.5 mg/kg bw ISMM in trypanosome-positive cattle [3,27]. Animals were grouped, ear-tagged and their weights estimated using a weighing band and conversion tables developed for local zebu cattle [28]. Group one was treated with Isometamidium chloride (TRYPAMIDIUM-SAMORIN ® , Merial, France) at the dose of 0.5mg/kg /bw (2% solution) and group 2 was treated with diminazene aceturate (DIM) (Veriben ® , Ceva Animal Health Inc., France) at 3.5 mg/kg bw (7% solution) against T. congulense. The preparations were injected deep intramuscular (i.m). Animals were monitored for trypanosomes and Paced Cell Volume (PCV) on day 14 and 28 post-treatment for ISMM and DIM. Those cattle failed to respond for each treatment were re-treated by shifting the drugs and were monitored for trypanosomes at day 14 and 28 post-treatment.

Sample collection and laboratory analysis
A jugular vein blood sample was extracted from every cattle into vacutainer tubes coated with di-sodium salt of ethylene diamine tetra-acetate (EDTA) using 18 gage needle and Hawksley micro haematocrit reader (Hawksley, Lancing, United Kingdom) was performed on blood in a capillary tube to determine the PCV whereas trypanosomes were detected using the dark-ground phasemicroscopy.

Data analysis
All data were coded and entered into excel spread sheet. Descriptive, Pearson chi square (χ2) and student's t-test analyses were carried out using SPSS (IBM SPSS Statistics for Windows, Version 20.0. IBM Corp, Armonk, NY.). Fisher exact test compared treatment failure between villages while Student's ttest was used to deter-mine differences in PCV values. Pearsonchi square (χ2) used to test differences in trypanosome prevalence between villages. The differences were considered statistically signi cant at P < 0.05.

Results
Overall prevalence of trypanosomosis The current study revealed that an overall prevalence of trypanosomosis was 4.50% (95% CI=4. 28, 4.72) in the studied areas. The prevalence of the disease was signi cantly different among districts (P<0.05 and the highest prevalence (7.1%) was recorded in Abeshege district. However, the difference was not signi cant (P>0.05) for sex, age and body condition ( Table 1) Evaluation paced cell volume for normal and infected cattle   showing treatment failure (n=11) at day 14 post treatment for isometamedium chloride were re-treated with diminazene aceturate (3.5 mg/kg/bw) and the failure rate was 54.5% (6/11) at 28 th days of post treatment ( Table 5). The highest and lowest treatment failure was recorded in Borer-5 50% (3/6)) and Misreta, Wolaita and Borer-4 of 16.7%1(1/6)) respectively.   (Table 7) and there was no signi cant difference amongst villages on treatment failure (P> 0.05).     [37] in different part of the country. The lower prevalence during this study might be due to difference in season of sampling and/or changes in ecological/ climatic conditions that affect vector y density/distribution overtime, animal susceptibility, trypanocidal drug use and existence of y control which affects the epidemiology of the disease [39][40] Eighty (n=80) known infected cattle were used to investigate the susceptibility T. congolense to two trypanocidal drugs namely DIM and ISM at different dose at day 14 and 28 post treatment. Accordingly, the study revealed that there was variability for failure rates at day 14 as compared to s at day 28 post treatment which was in agreement with other ndings in different parts of the country. For instance, the dynamic nature of the epidemiology of drug resistant infection in the Ghibe valley, which was reported to be 6% in 1986 and in1989, is increased to 14% [22]. The current nding showed that the incidence of multi drug resistance was increasing to 51.25% which was almost four fold increment as compared to the incidence in 1989.
Afework et al. [41] detected for the presence of resistance in mice using clones of T. congolense against both diminazene and isometamidium. According to Chaka and Abebe [3] T. congolense were found to be resistant to the curative action of diminazene (in mice and cattle) and isometamidium (in cattle) at a dose rate of 3.5 and 0.5 mg/kg body weight, respectively. Afewerk et al. [41] also showed the presence of multiple-drug-resistant T. congolense in the village cattle of Metekel district, northwest Ethiopia. Codjia et al. [18] reported thatonly one out of 12 trypanosome isolates collected from cattle in Ghibe were sensitive to 0.5 mg/kg b.w. isometamidium chloride, all were found to be resistant to 7.0 mg/kg, 0.5 mg/kg and 1.0 mg/kg body weight of diminazene, isometamidium and homidium chloride, respectively.
An overall failure rate for DIM in this nding was 47.5% (19/40) at 14 days post infection. The observed 3.5 mg/kg/bw DIM resistance is agreement with Afework et al. [41] had been reported the same failure rate in Ethiopia. Moreover, twenty percent (20%) failure rate was recorded to ISM during this study. Since long time ISMM resistance T.congolense has been reported in Ethiopia [42][43]. The multiple-drug resistance in the Ghibe valley of Ethiopia was related intensive use of quinapyramine sulphate for treating trypanosomiases [41]. The prolonged use of drugs without replacement, as is true for trypanocides principally encourages the development of resistance in accordance with Waller [44].

Conclusion
In conclusion, despite the low prevalence of trypanosomosis in the study areas, results from the current study revealed that T.congulonse developed multiple drug resistance against diminazene aceturate and isometamidium chloride. This is a serious impediment for controlling the disease in the areas. Therefore, the control of trypanosomosis in the study area should be quite rational in utilizing the available drugs and implementing an integrated control approach against the parasite and the vector.

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