Cattle Breeding, Trypanosomosis Prevalence and Drug Resistance in Northern Togo (2017)

Cattle breeding, trypanosomosis prevalence and drug resistance in Northern Togo

E. Tchamdja1, A.E. Kulo2, H.S. Vitouley 3, K. Batawui1, A.A. Bankolé1, K. Adomefa1, G. Cecchi4, A. Hoppenheit5, P. H. Clausen5, R. De Deken6, J. Van Den Abbeele7, T. Marcotty8, V. Delespaux9 *

1 Direction de l’Elevage, BP 4041, Lomé- Togo. 2 Ecole Supérieure d’Agronomie. Université de Lomé. BP 1515, Lomé –Togo. 3 Centre International de Recherche-Développement sur l’Elevage en Zone Subhumide (CIRDES), 01BP454 Bobo Dioulasso 01, Burkina-Faso. 4 Food and Agriculture Organization of the United Nations, Sub- Regional Office for Eastern Africa, Addis Ababa, Ethiopia. 5 Freie Universitaet Berlin, Institute of Parasitology and Tropical Veterinary Medicine, Robert-von-Ostertagstr. 7-13, 14163 Berlin, Germany. 6 Institute of Tropical Medicine, Biomedical Sciences Department, Veterinary Entomology, Nationalestraat 155, B-2000 Antwerp, Belgium. 7 Institute of Tropical Medicine, Biomedical Sciences Department, Veterinary Protozoology, Nationalestraat 155, B-2000 Antwerp, Belgium. 8 Veterinary Epidemiology, Risk-analysis and Diagnosis Research & Development, VERDI - R&D (asbl), Rue du gravier, 7, B-4141 Louveigné, Belgium. 9 Vrije Universiteit Brussel, Faculty of Sciences and Bioengineering Sciences, Pleinlaan 2 B-1050 Brussels, Belgium. *Corresponding author: Tel: +32-2-629 19 52; E-mail: [email protected]

The published journal article is available at: https://doi.org/10.1016/j.vetpar.2017.02.008

Abstract African Animal Trypanosomosis (AAT) is a major disease of cattle in Togo and its control is essentially based on chemotherapy. However, because of excessive use of trypanocides during the past decades, chemo-resistance in the parasites has developed. In order to assess the current situation of AAT and resistance to trypanocidal drugs in Northern Togo, a study was conducted on cattle from December 2012 to August 2013 in the regions of Kara and Savanes. An initial cross-sectional survey was carried out in 40 villages using the Haematocrit Centrifugation Technique (HCT). Out of these, 5 villages with a trypanosome prevalence of > 10% were selected for a block treatment study (BT) with diminazene diaceturate (DA: 3.5 mg / kg for a 14-day follow-up) and isometamidium chloride (ISM: 0.5 mg / kg for a 28-day follow-up). Positive blood samples collected during the parasitological surveys and an equivalent number of negatives were further analyzed by PCR-RFLP for trypanosome species confirmation and molecular diagnosis of resistance to DA in T. congolense. The results from 1,883 bovine blood samples confirmed a high overall trypanosome prevalence of 10.8% in Northern Togo. PCR-RFLP revealed that T. congolense is the dominant pathogenic trypanosome species (50.5%) followed by T. vivax (27.3%), and T. brucei (16.2%). The BT showed varying levels of treatment failures ranging from 0 to 30% and from 0 to 50% for DA and for ISM respectively, suggesting the existence of resistant trypanosome populations in the study area. Our results show that AAT still represents a major obstacle to the development of cattle husbandry in Northern Togo. In areas of high AAT risk, a community-based integrated strategy combining vector control, rational use of trypanocidal drugs and improving the general condition of the animals is recommended to decision makers.

Highlights

 African Animal Trypanosomosis is highly prevalent in Northern Togo  Trypanocidal drug resistance is widespread  Community based integrated control strategy is recommended

Keywords African Trypanosomosis, chemo-resistance, trypanocides, block treatment, PCR-RFLP, Northern Togo. Introduction In sub-Saharan Africa, African Animal Trypanosomosis (AAT) remains one of the main constraints to the development of the livestock sector and of crop-livestock mixed farming. AAT has slowed overall structural development in Africa by preventing growth of human settlements through a lack of suitable animals for transportation and farming (Alsan 2015). Today, more than 55 million head of cattle are at risk of the disease. The trypanosome parasites causing AAT (Trypanosoma congolense, T. vivax, T. brucei brucei) are mainly transmitted by hematophagous tsetse flies (Glossina sp.) that are spread over an area of about 10 million square kilometers (Cecchi and Mattioli 2009).

In Togo, AAT is enzootic (Mawuena and Yacnambe 1990; Hendrickx et al. 1999; Dao et al. 2008; Talaki et al. 2014) and interventions against the disease are mainly executed by farmers who rely on trypanocidal drugs, i.e. the curative diminazene diaceturate (DA) and the preventive isometamidium chloride (ISM). Increased development of resistance in the parasites, oftentimes to both trypanocides, was reported in several east- and west-African countries by Delespaux et al. (2008) and Clausen et al. (2010), respectively. In Togo resistance was observed by Vitouley et al. (2013). Another constraint to a successful chemotherapy is the oftentimes insufficient quality of the available trypanocides which has been described for Togo by Tchamdja et al. (2016). Thus, knowledge of such key epidemiological factors as disease prevalence and trypanocidal resistances is needed to develop integrated strategies for communities in areas with high AAT risk (Mungube et al. 2012a; Shaw et al. 2015).

Various methodologies for the detection of treatment failures and the diagnosis of resistance to trypanocidal drugs were reviewed by Delespaux et al. (2008). Among these methods, the most commonly used are: (i) follow-up of the persistence of trypanosome infections in animals treated with trypanocidal drugs, called ''block treatment'' (BT) (Eisler et al. 2000; Mungube et al. 2012b); (ii) in vivo standardized drug sensitivity tests on isolated field-strains in mice or cattle (Eisler et al. 2001) and (iii) molecular tools (Delespaux et al. 2008). For detecting resistance, field tests (BT) and laboratory tests (in vivo tests) are based on the detection of trypanosomes after secundum artem administration of a trypanocide, while molecular tools seek to identify genetic markers associated with a phenotype of drug resistance. Currently, markers of resistance are available for DA but up to date no reliable marker has been developed to diagnose ISM-resistance (Mamoudou et al. 2008; Delespaux et al. 2008).

The EU-funded project "Trypanosomosis Rational Chemotherapy" (TRYRAC) was implemented in three African countries (Ethiopia, Mozambique and Togo) with the main objective of improving the livelihoods of low-income farmers through a more efficient use of trypanocides in AAT enzootic areas. As part of the project, the present study was conducted in northern Togo (regions of Kara and Savanes) to generate up-to-date information on the occurrence of AAT in the region, and to fill the knowledge gap on drug use and drug resistance. The study focused on: (i) determining trypanosome prevalence and species through a cross-sectional survey, (ii) assessing the levels of treatment failures to trypanocides by BT in villages with a high AAT prevalence, and (iii) designing and implementing the most adequate strategy to control the disease. Materials and methods Study area The cross-sectional study was conducted from December 2012 to February 2013 in the regions of Kara and Savanes in Northern Togo. The area (20,208 square kilometres) corresponds to 35% of the total surface of the country, and it hosts a large part of the national cattle population (71%, i.e. approximately 150,000 head) (Ministère de l’Agriculture Elevage et Pêche 2013).The two main cattle breeds in the Savanes and Kara regions are the Sahelian zebu and the Somba taurine. Togo has a tropical savannah climate which is characterized by one single rainy season from April to October and a dry season from November to March. AAT is endemic and caused by T. congolense, T. vivax and T. brucei in different proportions depending on the regions. The main vectors of these trypanosomes are Glossina morsitans submorsistans, Glossina palpalis palpalis and Glossina tachinoides (Hendrickx et al. 1999; Dao et al. 2008; Talaki et al. 2014).

Based on public domain geospatial databases (Cecchi and Mattioli 2009; Cecchi et al. 2014), aerial maps, field visits, livestock census and expert opinions, 71 villages were initially identified as at-risk for trypanosomosis. Other criteria had to be met as well, including high cattle density, presence of dense vegetation suitable for tsetse (Cecchi et al. 2008), presence of rivers and clinical reports of the disease by veterinarians. Out of the 71 villages, 40 were randomly selected (Figure 1). Cattle herds were identified by drawing data from the local Veterinary Services census. Sampling framework Independently of the size of the herds, 50 cattle were sampled in each of the 40 villages to detect at least one positive, with 90% certainty and assuming the AAT prevalence is more than 5% (Thrusfield 1995). Parasitological analysis: Haematocrit Centrifugation Technique (HCT) Blood was collected from the jugular vein in EDTA BD Vacutainer® tubes (Becton-Dickinson, Franklin Lakes, New Jersey, USA), transferred to capillary tubes and sealed with Cristaseal® (Hawksley, Sussex, UK). Tubes were centrifuged at 13,700 g for 5 minutes. The buffy coat was examined for the presence of trypanosomes as described by Woo (1970). Trypanosome species were identified by size and movement pattern. In addition, for each selected animal, the packed cell volumes (PCV) as well as the sampling location, owner’s name, age, sex, breed and body condition were recorded.

Figure 1 Map of the study area showing the cross-sectional survey villages and sites of Northern Togo where the block treatment was carried out. Information on drug use Farmers, private veterinarians and heads of veterinary posts of the 40 villages were interviewed about the frequency of trypanocidal treatments. Three categories were defined as follows: "high" for more than 3 treatments per year, "low" for less than 2 times and “average” for 2-3 treatments (Van den Bossche et al. 2000). Block treatment The block treatment was conducted in five villages (Figure 1) from June to August 2013. The criterion for inclusion in the BT was a high trypanosome prevalence in the village cattle herds (10 percent or more). Three villages were selected for a drug use frequency reported as high and two as low. In each selected village, 20 trypanosome-positive animals were randomly selected and then divided into two groups of 10 animals each, as described by Mungube et al. (2012). Animals were ear-tagged and their body weight was estimated by thoracic perimeter and the conversion tables of Buldgen et al. (1984) and Adanléhoussi et al. (2003), suitable for local Somba taurine and zebu or zebu crossbreeds respectively.

The first group was treated with DA (Trypadim® by Merial, Lyon, France) at 3.5 mg/kg b. w. of a 7% solution. The second group was treated with ISM (Trypamidium®by Merial, Lyon, France) at 0.5 mg/kg b.w. of a 1% solution. Prior to their use, the trypanocides were analysed by HPLC in order to assure their compliance with OIE standards (Tchamdja et al. 2016). The animals were monitored for their PCV and trypanosomal infection on the day of treatment (D0) and at day 14 (D14) and at day (D28) after treatment. Positive animals at D14 and/or D28 were treated with DA at 7 mg/kg b. w.

Blood processing for molecular analysis Approximately 0.75 ml of blood was sampled from each HCT-positive animal. Samples were preserved in an equal volume of saturated 6 M guanidine buffer and stored at -18°C for further molecular analysis. For each HCT-positive sample, a negative sample was randomly selected out of the pool of the HCT-negative samples. A total of 554 samples were analyzed: 354 samples (177 positive and 177 negative) from the cross-sectional study and 200 samples (100 positive and 100 negative), from the block treatment study. DNA extraction and 18S-PCR-RFLP screening DNA was extracted from 200μl of the blood/guanidine buffer mixture using the commercial kit QIAamp® DNA Blood Mini kit (Qiagen Inc., Valencia, California, USA). For trypanosome species confirmation, DNA amplifications were done using three primers which target the 18s small ribosomal subunit in a semi-nested protocol, followed by restriction of the amplicons by Msp1 and Eco571 enzymes (New England BioLabs, Ipswich, Massachusetts, USA) as described by Geysen et al. (2003). Diminazene diaceturate DpnII-PCR-RFLP resistance test All T. congolense-positive samples (based on the 18S-PCR-RFLP test) were amplified using two primers targeting the P1-type purine transporter TcoNT10 gene. Then, the PCR products were restricted with the DpnII enzyme for DA resistance determination (Delespaux et al. 2006; Vitouley et al. 2011; Delespaux and de Koning 2013; Munday et al. 2013). Data analysis Robust logistic regression was used to analyse data on prevalence and treatment failures. Clusters were villages and cattle owners in the cross-sectional and BT studies, respectively. Sampling probabilities resulting from different herd sizes were ignored. A backward- selection of estimators was applied on cross-sectional data to identify significant explanatory variables (region, age and sex) using a threshold of 10%. Similarly, PCV was analyzed by means of linear regression using the same clusters and the same explanatory variables. In the BT study, the significance of region and drug use frequency on treatment failure was evaluated in univariate models, separately for DA and ISM. DA treatment failure was recorded when trypanosomes (T. vivax or T. congolense) were found in animals on day 14 after DA treatment. ISM treatment failure was recorded when trypanosomes (T. vivax or T. congolense) were found in animals on days 14 or 28 after ISM treatment. Considering the low prevalence and the low pathogenicity of T. brucei in cattle, this species was not further considered in the BT analysis. Compliance with ethical standards The experimental protocol and sampling were approved by the Ethical Committee of the University of Lomé. The background of the study was explained to the peasant associations and consent of the farmers was requested for the sampling of their animals. Results Cross-sectional parasitological survey Within the study area, 1,883 blood samples were collected from an estimated cattle population of 15,714 in 254 herds. Ninety livestock breeders responded on their use of trypanocides. The trypanosome prevalence, the average PCV, and drug use intensity are

Figure 2 Prevalence of AAT (A), PCV (B) and intensity of drug use (C) in the study area.

Assuming the sampling fraction and the prevalence to be independent, the overall prevalence was estimated at 10.2%. The average prevalence of the Savanes region (12.6%) was higher than that of the Kara region (8.8%), but no statistical significance was observed in a robust model (p = 0.13). The average PCV in both Kara and the Savanes regions were 24.1 ± 0.8 and 24.5 ± 0.9, respectively (average ± robust standard error; p = 0.47). It was observed that the average PCV of trypanosome-positive animals (22.8 ± 1.0) was significantly lower than that of trypanosome-negative animals (24.4 ± 0.6) (p = 0.001).

Significantly more young animals (<2 years) were infected (13.23 %) compared to adults (8.7 %) (P = 0.045). Independently from infection, young animals showed lower PCV’s (23.8 ± 0.3) than adults (24.7 ± 0.3) (average of non-infected animals ± robust standard error; P = 0.003).

T. congolense and T. vivax were the two most commonly found trypanosome species with 93.3% of all single and mixed infections, whereby 6.7% of the detected trypanosomes could not be identified by microscopy. Those unidentified trypanosomes could be either T. congolense, T. vivax or T. brucei with unclear movement pattern and/or visibility within the microscopic preparation.

Koundoum (14.0% prevalence) in Kara region, Magna (26.0%) and Kadjitiéri (14.0%) in Savanes region were identified as villages with high trypanosome prevalence and high trypanocidal drug use. Wakadè (24.0%) in Kara region and Lopano (28.0%) in Savanes region were identified as sites with high prevalence and low drug use (Tables 1 and 2). Treatment response to diminazene Treatment with DA (3.5kg/kg) revealed a failure rate of 14% on day 14 (Table 3). In Kara region this rate was 15.0%, as compared to 13.3% in the Savanes region (no significant difference, P=0.89). The villages with high trypanocidal drug use showed a failure rate of 20.0% compared to 5% for the villages who reported average / low use (p=0.2). Second DA treatments at a dose of 7 mg/kg failed to cure in 42.9% of cases of relapsing trypanosomosis. Treatment response to isometamidium The results of treatment with ISM are shown in Table 3. The BT performed on 50 animals showed a failure rate of 26.0% (cumulative on days 14 and 28 post-treatment). Kara region presented a failure rate of 30.0% compared to 23.3% for Savanes region (p=0.65). Villages with a high use of trypanocides showed a failure rate of 36.0% while the rate was 10.0% in villages with an average / low use (p=0.08). Results of MspI-PCR-RFLP The MspI-PCR-RFLP protocol applied to samples from the cross-sectional survey allowed the diagnosis of three species of pathogenic trypanosomes in single infections (T. congolense, T. vivax and T. brucei) or mixed ones (T. congolense / T. vivax and T. congolense / T. brucei). The non-pathogenic T. theileri was also found, either in single infections or associated with pathogenic trypanosomes. Table 4 shows that T. congolense was the dominant species throughout the study area (50.5% of infections in total; 53.2% in Kara), while T. vivax was dominant in Savanes with 50.0% of infections. The data of table 4 displayed per village is available as Electronic Supplementary Material. Results of the DpnII-PCR-RFLP Out of 56 tested T. congolense infections (single and mixed), 34 gave a positive DpnII-PCR- RFLP result, reflecting a DA-resistant profile. Out of these, 46.7% originated from BT isolates after DA treatment failures. Discussion The present study was conducted in the Kara and Savanes regions to confirm the persistence of AAT in the main livestock production areas in Togo, and to link this information with the intensity of drug use and drug resistance.

When comparing the average trypanosome prevalence of 10.3% (by HCT) in this study to the 1.0 to 12.0% reported by Hendrickx et al. (1999), it appears that trypanosomosis remains a major constraint to the Togolese livestock sector. As expected, a significant difference in PCV (1.6 ± 0.45 percent) was observed between trypanosome - positive and negative animals (Marcotty et al. 2008).

T. congolense and T. vivax were the two trypanosome species identified by microscopy, confirming similar observations in the same region (Hendrickx et al. 1999; Dao et al. 2008). However, three species were identified by Talaki et al. (2014), namely T. congolense, T. vivax

Cattle breeding, trypanosomosis prevalence and drug resistance in Northern Togo (2017). Tchamdja E., Kulo A.E., Vitouley H.S., Batawui K., Bankole A.A., Adomefa K., Cecchi G., Hoppenheit A.; Clausen P.H.; De Deken R., Van Den Abbeele J., Marcotty T., Delespaux V. Veterinary Parasitology, 236:86–92. and T. brucei with T. vivax as predominant species. In this study, PCR analyses confirmed the presence of these three species of pathogenic trypanosomes but with an overall predominance of T. congolense although T. vivax was the dominant species in Savanes as shown by the PCR results (Table 4). This observation could be related to the role of mechanical vectors in this area (Desquesnes and Dia 2003; Desquesnes and Dia 2004) and a low perceived density of tsetse flies (Hoppenheit et al. 2014) due to human encroachment and drought that destroyed their habitat in the Savanes region Dao et al. (2008).

The field technique commonly used for the diagnosis of resistance to trypanocides (Eisler et al. 2000; Mamoudou et al. 2008) is laborious, expensive and especially burdensome for farmers, with the recommended follow-up period of 60 or 90 days. The shortened BT protocol of 28 days which was applied in this study has the advantage of quickly generating results without being too cumbersome for herders and technicians, resulting in an improved cost-benefit ratio (Mungube et al., 2012). A disadvantage, however, is the low sensitivity of the applied HCT method for the diagnosis ranging between 300-700 trypanosomes per ml of blood (Uilenberg 1998). This short BT is thus limited by its inability to detect infections with very low parasitaemia (Woo an d Rogers, 1974; Desquesnes and de la Rocque, 1995) and may lead to an underestimation of treatment failures. Moreover, it does not provide information on the intensity of infected tsetse challenge as negative controls (untreated herds) are not included in the study, for ethical reasons.

The sensitivity of trypanosome to DA in the BT studies showed varying rates of treatment failures among the tested village herds. These observed treatment failures are likely to be linked to the presence of resistant strains of trypanosomes. Indeed, it has been observed that a significant proportion of trypanosomes which were not eliminated at the dose of 3.5 mg DA/kg b.w. survived at the dose of 7 mg/kg b.w. Similar results were previously reported in several studies with trypanosomes surviving DA doses up to 17.5 mg/kg (Clausen et al. 1992).

Trypanosome sensitivity to ISM by BT also showed varying rates of treatment failures between the geographical localizations. Normally, the concept of the sanative pair recommends the use of two trypanocides (e.g. DA and ISM) unlikely to induce cross- resistance. The first drug is used until resistant strains of trypanosomes appear, and then it is substituted by the second drug until trypanosomes cannot be detected in cattle and tsetse (Whiteside 1962). In this study, persisting trypanosome-infections following ISM treatment were eliminated by the administration of DA at 7 mg/kg b.w. However, this cannot be interpreted as an efficient sanative pair intervention because at 14 days after administration of the DA or 28 days after ISM administration the plasma concentration of ISM is still high enough to influence the action of the second drug (Eisler et al., 1997; Mungube et al., 2012).

The results of the BT showed a higher treatment failure for ISM compared to DA. The same observations were reported by Geerts et al., (2001); McDermott et al., (2003); Sow et al., (2012) and Mungube et al., (2012). This could be explained by the pharmacokinetic of ISM which results in an extended exposure of trypanosomes to the active ingredient before its elimination from the system (Eisler et al. 1997).

Although no statistically significant difference was observed in sensitivity between villages with frequent versus less frequent trypanocidal use, a tendency towards increased treatment failures in villages with frequent trypanocidal use was noticed, confirming the positive correlation between highly frequent trypanocidal use and the presence of drug resistance (Geerts and Holmes 1998; Delespaux and de Koning 2013).

Molecular tools are alternatives to the commonly used field tests in the diagnosis of trypanocidal drug resistance. These tests are more sensitive, less expensive and they can be performed on a large number of samples (Delespaux et al. 2008). However, the rate of mutated alleles in the TcoNT10 P1-type purine transporter gene used as marker reaches systematically 90-100% in areas of frequent drug use as observed in several field studies (Delespaux and de Koning 2013). Observations of this study in Northern Togo corroborate these results, as 100% of the tested trypanosomes showed resistant profiles in RFLP. The short BT thus remains a reliable reference tool for a quick and relatively cost-efficient estimation of drug resistance in the field, with an advantage that the molecular approach cannot provide, i.e. a more quantitative estimate of the phenotypic drug resistance. Conclusions Our results show that (i) trypanosomosis remains a major constraint to animal husbandry in Northern Togo, (ii) the high trypanosome prevalence indicates an association with frequent treatment failures and (iii) cases of multiple resistances were observed in four villages, which compromise the effectiveness of the sanative pair (Whiteside 1962). As long as AAT management is considered the sole responsibility of the Togolese farmers, these results suggest that more successful disease interventions should be based on an integrated management strategy involving the affected communities, as recommended by Clausen et al. (2010) and Bardosh (2015). This integrated strategy involves: (i) a rational use of trypanocidal drugs, (ii) the control of vectors, (iii) boosting the immunity of animals at risk through strategic deworming at the beginning and at the end of the rainy season, as well as feed supplements during times of scarcity. In addition, a rigorous quality control of available trypanocides in Togo and the strengthening of qualified veterinary services in proximity to the herds would help to contain the further development and spread of trypanocidal resistances. Acknowledgments

The authors thank the European Union for funding this study through the EU funded TRYRAC project (DCI-FOOD/2011/279-754). Their thanks also go to CIRDES for PCR analyses performed as part of this work.

FAO assistance to this study was provided in the framework of the Programme Against African Trypanosomosis (PAAT), and supported by the Government of Italy (Project ‘Improving food security in sub-Saharan Africa by supporting the progressive reduction of tsetse-transmitted trypanosomosis in the framework of the NEPAD’, codes GTFS/RAF/474/ITA and GCP/RAF/502/ITA).

References Adanléhoussi A, Bassowa H, Défly A, et al (2003) Les performances de la race taurine Somba en milieu paysan. Tropicultura 3:135–141. Alsan M (2015) The effect of the tsetse fly on African development. Am Econ Rev 105:382–410. doi: 10.1257/aer.20130604 Bardosh KL (2015) Deadly flies, poor profits and veterinary pharmaceuticals: sustaining the control of sleeping sickness in Uganda. Med Anthropol. doi: 10.1080/01459740.2015.1101461 Buldgen A, Compère R, Riboux A (1984) Recherche d’une formule barymétrique adaptée aux bovins de type Djakoré dans les élevages villageois du Sénégal Oriental. Tropicultura 2:86 – 90. Cecchi G, Mattioli RC (2009) Global geospatial datasets for African trypanosomiasis management: a review. In: Cecchi G, Mattioli RC (eds) Geospatial datasets and analyses for an environmental approach to African trypanosomiasis. Food and Agriculture Organization of the United Nations, Rome, pp 1–39 Cecchi G, Mattioli RC, Slingenbergh J, De La Rocque S (2008) Land cover and tsetse fly distributions in sub-Saharan Africa. Med Vet Entomol 22:364–373. doi: 10.1111/j.1365-2915.2008.00747.x Cecchi G, Paone M, Feldmann U, et al (2014) Assembling a geospatial database of tsetse-transmitted animal trypanosomosis for Africa. Parasit Vectors 7:39. doi: 10.1186/1756-3305-7-39 Clausen P-H, Bauer B, Zessin KH, et al (2010) Preventing and containing trypanocide resistance in the cotton zone of West Africa. Transbound Emerg Dis 57:28–32. Clausen P-H, Sidibe I, Kabore I, Bauer B (1992) Development of multiple drug resistance of Trypanosoma congolense in zebu cattle under high natural tsetse fly challenge in the pastoral zone of Samorogouan, Burkina Faso. Acta Trop 51:229–236. Dao B, Hendrickx G, Sidibé I, et al (2008) Impact de la sécheresse et de la dégradation des aires protégées sur la répartition des trypanosomoses bovines et de leurs vecteurs dans le bassin versant de l ’ Oti au nord du Togo. Rev d Elev Med Vet des Pays Trop 61:153 – 160. Delespaux V, Chitanga S, Geysen D, et al (2006) SSCP analysis of the P2 purine transporter TcoAT1 gene of Trypanosoma congolense leads to a simple PCR-RFLP test allowing the rapid identification of diminazene resistant stocks. Acta Trop. 100:96–102. Delespaux V, de Koning HP (2013) Transporters in anti-parasitic drug development and resistance. In: Jäger T, Och O., Flohé L (eds) Trypanosomatid Diseases: Molecular Routes to Drug Discovery. Wiley-VCH Verlag GmbH & Co. KGaA, pp 335–349 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. doi: 10.1016/j.pt.2008.02.006 Desquesnes M, de la Rocque S (1995) Comparison of the sensitivity of the Woo test and a test for detecting antigens to Trypanosoma vivax in 2 sheep experimentally infected with a Guyanese strain of the parasite. Rev d Elev Med Vet des Pays Trop 48:247–253. Desquesnes M, Dia ML (2003) Mechanical transmission of Trypanosoma congolense in cattle by the African tabanid Atylotus agrestis. Exp Parasitol 103:35–43. Desquesnes M, Dia ML (2004) Mechanical transmission of Trypanosoma vivax in cattle by the African tabanid Atylotus fuscipes. Vet Parasitol 119:9–19. Eisler MC, Brandt J, Bauer B, 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.

Eisler MC, Gault E. A, Moloo S. K, et al (1997) Concentrations of isometamidium in the sera of cattle challenged with drug-resistant Trypanosoma congolense. Acta Trop 63:89–100. doi: 10.1016/S0001-706X(96)00602-X Eisler MC, McDermott JJ, Mdachi RE, et al (2000) Rapid method for the assessment of trypanocidal drug resistance in the field. Proc 9th Symp Int Soc Vet Epidemiol Econ Nairobi, Pap 353 9:1–3. Geerts S, Holmes PH (1998) Drug management and parasite resistance in bovine trypanosomiasis in Africa. PAAT Tech. Sci. Ser. 1:-. Geerts S, Holmes PH, Diall O, et al (2001) African bovine trypanosomiasis : the problem of drug resistance. Trends Parasitol 17:18–21. Geysen D, Delespaux V, Geerts S (2003) PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle. Vet. Parasitol. 110:171–180. Hendrickx G, Napala A, Dao B, et al (1999) The area-wide epidemiology of bovine trypanosomosis and its impact on mixed farming in subhumid West Africa; a case study in Togo. Vet Parasitol 84:13–31. Hoppenheit A, Bauer B, Delespaux V (2014) Fact Finding Mission to Togo, September 30-October 11 2013, EU-funded project: Trypanosomosis Rational Chemotherapy (TRYRAC), http://www.trypanocide.eu/partner-area/reports/mission- reports/,Fact_Finding_Mission_Togo_FUB_2013.pdf [1.2 MB]. 1 – 33. Mamoudou A, Delespaux V, Chepnda V, et al (2008) Assessment of the occurrence of trypanocidal drug resistance in trypanosomes of naturally infected cattle in the Adamaoua region of Cameroon using the standard mouse test and molecular tools. Acta Trop. 106:115–118. Marcotty T, Simukoko H, Berkvens D, et al (2008) Evaluating the use of packed cell volume as an indicator of trypanosomal infections in cattle in eastern Zambia 2. Prev Vet Med 87:288–300. Mawuena K, Yacnambe MS (1990) Emploi de pièges et d’écrans dans la lutte contre la trypanosomiase animale au Togo. Tropicultura 8:40 – 43. McDermott JJ, Woitag T, Sidibe I, et al (2003) Field studies of drug-resistant cattle trypanosomes in Kenedougou Province, Burkina Faso. Acta Trop. 86:93–103. Ministère de l’Agriculture Elevage et Pêche (MAEP) (2013) Profil de l’agriculture togolaise. In: 4ème recensement national de l’agriculture 2011-2014. Ministère de l'Agriculture, de l'Elevage et de la Pêche(MAEP).Direction des Statistiques Agricoles ,de l'Information et de la Documentation. p 33 Munday JC, Rojas López KE, Eze AA, et al (2013) Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake. Int. J. Parasitol. Drugs drug Resist. Mungube EO, Diall O, Baumann MPO, et al (2012a) Best-bet integrated strategies for containing drug-resistant trypanosomes in cattle. Parasit. Vectors 5:164, doi:10.1186/1756–3305–5–164. Mungube EO, Vitouley HS, Allegye-Cudjoe E, et al (2012b) Detection of multiple drug-resistant Trypanosoma congolense populations in village cattle of south-east Mali. Parasit Vectors 5:155. doi: 10.1186/1756-3305-5-155 Shaw APMPM, Wint GRW, Cecchi G, et al (2015) Mapping the benefit-cost ratios of interventions against bovine trypanosomosis in Eastern Africa. Prev Vet Med 122:1–11. doi: 10.1016/j.prevetmed.2015.06.013 Sow A, Sidibe I, Bengaly Z, et al (2012) Field detection of resistance to isometamidium chloride and diminazene aceturate in Trypanosoma vivax from the Region of the Boucle du Mouhoun in Burkina Faso. Vet Parasitol 187:105–111.

Talaki E, Dayo GK, Akoda K, et al (2014) Epidemiology of Bovine Trypanosomosis in Savannah and Kara Regions in Northern Togo. Int Invent J Agric Soil Sci 2:126–131. Tchamdja E, Akoda K, Kulo AE, et al (2016) Drug quality analysis through high performance liquid chromatography of isometamidium chloride and diminazene aceturate purchased from official and unofficial sources in Northern Togo. Prev Vet Med 126:151–158. doi: 10.1016/j.prevetmed.2016.02.001 Thrusfield M (1995) Veterinary Epidemiology, 2nd edn. Blackwell Science, Oxford, U.K., ISBN-10: 1- 405-1562. Van den Bossche P, Doran M, Connor RJ (2000) An analysis of trypanocidal drug use in the Eastern Province of Zambia. Acta Trop 75:247–258. Vitouley HS, Bengaly Z, Adakal H, et al (2013) Réseau d’épidémio-surveillance de la chimiorésistance aux trypanocides et aux acaricides en Afrique de l’Ouest (RESCAO). Tropicultura 85:205–212. Vitouley HS, Mungube EO, Allegye-Cudjoe E, et al (2011) Improved PCR-RFLP for the detection of diminazene resistance in Trypanosoma congolense under field conditions using filter papers for sample storage. PLoS Negl Trop Dis 5:7–e1223 doi:10.1371/journal.pntd.0001223. Whiteside EF (1962) The control of cattle trypanosomiasis with drugs in Kenya: Methods and costs. East AfrAgrJ 28:67–73. Woo PTK (1970) The haematocrit centrifuge technique for the diagnosis of African trypanosomiasis. Acta Trop 27:384–387. Woo PTK, Rogers DJ (1974) A statistical study of the sensitivity of the haematocrit centrifuge technique in the detection of trypanosomes in blood. Trans R Soc Trop Med Hyg 68:319–326.

Tables Table 1. Trypanosome prevalence (%), average PCV (%) and drug use intensity of Kara region, June 2013

Villages N PCV N+ Tc Tv Tc/Tv T.sp Prev. DU Agbassa 50 26.2 ±1.3 7 - 6 1 14,0 ++

Bidjandè 45 28.6 ±1.6 4 3 - 1 - 8.9 + Bounoh 50 21.2 ±1.00 1 1 - - - 2,0 ++ Broukou 50 26.7 ±1.3 3 2 - - 1 6,0 ++ Dandare 50 26.4 ±1.3 7 6 - - 1 14,0 + Delabre 50 25.6 ±0.9 2 2 - - - 4,0 + Kawa 50 25.4 ±1.3 1 1 - - - 2,0 + Hélota 50 24.6 ±1.0 0 0 - - - 0,0 + Konkouboune 44 22.1 ±1.1 5 5 - - - 11.4 + Koudjoudjou 50 21.9 ±1.0 8 8 - - - 16,0 + Koudjoukponkpon 50 23.0 ±1.0 13 13 - - - 26,0 + Koundoum* 50 22.6 ±1.2 7 7 - - - 14,0 +++ Koulintè 50 25.9 ±0.9 1 1 - - - 2,0 ++ Koutchoutchéou 40 23.6 ±1.0 0 0 - - - 0,0 + Koutière 18 26.6 ±1.9 0 0 - - - 0,0 + Kpatchile 50 20.7 ±1.0 4 4 - - - 8,0 + Kpessidè 37 24.5 ±1.6 6 6 - - - 16.2 + Langa 49 22.6 ±1.1 4 4 - - - 8.16 + M'Borotchika 50 22.5 ±1.0 1 0 1 - - 2,0 + Nanani 50 22.4 ±0.7 4 4 - - - 8,0 + Pangouda 50 23.5 ±1.0 2 2 - - - 4,0 + Tchoré 50 23.0 ±1.0 6 6 - - - 12,0 + Toguel 50 26.2 ±1.1 5 5 - - - 10,0 + Typoul 50 26.2 ±1.3 1 1 - - - 2,0 + Wakadè* 50 21.1 ±0.9 12 12 - - - 24,0 + Total 1,183 24.1±0.3 104 93 7 1 3 8.8

With N=Number of animals examined; PCV=Average PCV with 95% CI; N+=number of positive animals; Tc as T. congolense; Tv as T. vivax; DU= drug use: +++ high; ++ average ; + low ;* : Villages selected for BT

Table 2. Trypanosome prevalence (%), average PCV (%) and drug use intensity of Savanes region, June 2013

Villages N PCV N+ Tc Tv Tc/Tv T.sp Prev. DU Djapal 50 24.3 ±1.0 5 5 - - - 10.0 + Faré 50 23.6 ±1.0 6 5 - - 1 12.0 + Gando 50 25.0 ±1.1 1 1 - - - 2.0 +++ Poloti 50 28.1 ±1.3 4 1 - - 3 8.0 +++ Kerkete 50 22.5 ±1.1 7 7 - - - 14.0 ++ Koumongoukan 25 24.3 ±1.6 3 3 - - - 12.0 + Kpakpabou 30 23.3 ±1.4 3 1 - - 2 10.0 + Lopano* 50 25.5 ±1.1 14 14 - - - 28.0 + Magna* 50 21.8 ±1.3 13 13 - - - 26.0 +++ Nagouni 50 23.7 ±1.0 6 6 - - - 12.0 + Panion 45 26.2 ±1.4 1 1 - - - 2.22 + Kadjitiéri* 50 27.3 ±1.2 7 4 2 - 1 14.0 +++ Sadori 50 25.1 ±1.1 3 2 - - 1 6.0 ++ Santigou 50 22.6 ±0.9 11 5 4 - 2 22.0 ++ Takpamba 50 24.2 ±1.1 6 6 - - - 12.0 + Total 700 24.5 ±0.3 90 74 6 - 10 12.9

With N=Number of animals examined; PCV=Average PCV with 95% CI; N+=number of positive animals; Tc as T. congolense; Tv as T. vivax; Tc/Tv as mixed infection T. congolense and T. vivax; T. sp as Trypanosome infection not identified at species level; Prev. as prevalence; DU= drug use +++ high; ++ average ; + low ;* : Villages selected for BT

Table 3. Treatment failure rate (%) in trypanosome-positive cattle treated with DA (3.5 mg/kg b.w.) and treated with ISM (0.5 mg/kg b.w.)

Region Village N* TFR DA** TFR ISM*** Kara Koundoum 10 20.0% 40.0% Wakadè 10 10.0% 20.0%

Subtotal 20 15.0% 30.0% Savanes Lopano 10 0.0% 0.0% Magna 10 30.0% 50.0%

Kadjitiéri 10 10.0% 20.0%

Subtotal 30 13.0% 23.3% Total 50 4.0% 26.0%

With * number of treated cattle (10 with DA and 10 with ISM); ** treatment failure rate at day 14 after treatment for the DA-treated animals; *** cumulated treatment failure rate measured at day28 for the animals unsuccessfully treated on day 0 with 0.5 mg ISM/kg b.w. and on day 14 with 7 mg DA/kg b.w.

Table 4: Trypanosome species diagnosis by MspI-PCR-RFLP

Region Tested + Tc Tv Tb Tc/Tv Tc/Tb Kara 198 77 41 (53.2%) 16 (20.8%) 14 (18.2%) 2 (2.6%) 4 (5.2%) Savanes 156 22 9 (40.9%) 11 (50.0%) 2 (9.1%) - -

Total 354 99 50 (50.5%) 27 (27.3%) 16 (16.2%) 2 (2.0%) 4 (4.0%)

With + as PCR positive; Tc as T. congolense; Tv as T. vivax; Tb as T. brucei