Assessment of α-cypermethrin Pour-on Application and Diminazene Aceturate for the Treatment of Trypanosome-related Diseases Caused by Tsetse Flies in Cattle in Mô (Togo)

Soudah Boma (  bomasoudah@gmail.com ) International Center for Research and Development on Livestock Production in the Subhumid Zone: Centre International de Recherche-Developpement sur l'Elevage en zone Subhumide https://orcid.org/0000-0003-3862-5308 Essodina Talaki Université de Lomé ESA: Ecole Supérieure d'Agronomie Toï N'Féidé Togolese Institute for Agronomic Research: Institut Togolais de Recherche Agronomique Balabadi Dao Togolese Institute for Agronomic Research: Institut Togolais de Recherche Agronomique Yao Lombo Togolese Institute for Agronomic Research: Institut Togolais de Recherche Agronomique Martin Bienvenu Somda International Center for Research and Development on Livestock Production in the Subhumid Zone: Centre International de Recherche-Developpement sur l'Elevage en zone Subhumide Wendemanegdé Ernest Salou International Center for Research and Development on Livestock Production in the Subhumid Zone: Centre International de Recherche-Developpement sur l'Elevage en zone Subhumide


Abstract Background
The effects of tsetse-transmitted trypanosomosis control in high tsetse challenge and trypanocidal drug resistance settings remain poorly understood in Togo, owing to the poor data underlying the current disease impact. This study was implemented in the framework of the PDRI-Mô project which is an African animal trypanosomosis control project using trypanocides and insecticides on cattle. This project, planned by the agricultural ministry, focused on all the sedentary cattle breeds in the 1000 km² area of the prefecture of Mô. Until 2013, the locality remained unconnected to outside areas, with long-term limited access to quality medicines, veterinary services, animal husbandry and new AAT control technologies.

Methods
From March 2014 to November 2017, a database of zoo-sanitary surveys integrating the evolution of the incidence of the disease and the coverage of interventions made it possible to quantify the apparent effect attributable to control effort in herds. The strategy involved an initial phase with cross-sectional entomological and parasitological surveys, including a rapid trypanocidal drug sensitivity testing.
Treatment e cacy was assessed using parasitological status post-treatment of trypanosome-positive animals in each village, randomly assigned to three groups, one treated with 0.5 mg/kg b.w.
Isometamidium chloride, the second with 3.5 mg/kg b.w. Diminazene diaceturate and the third with distillated water as control. Using parasitological status of blood sample collected on day 0, day 14 and day 28 post-treatment as the outcome result, trypanosome phenotype resistance of drugs treatment was determined if relapse occur. Then, three times a year, 20% of the herd received α-cypermethrin pour-on, and parasitaemic cattle with poor health were individually given diminazene aceturate at 7 mg/kg of body weight (b.w.).

Results
The tsetse density in the area decreased signi cantly (P-value 0.001) from 1.78 ± 0.37 in March 2014 prior to the α-cypermethrin application to 0.48 ± 0.07 in February 2017. Prior to the trypanocidal treatment, relapse of trypanosome infections in the cattle was approximately 12.5% for diminazene at 3.5 mg/kg of b.w., 35% for isometamidium at 0.5 mg/kg of b.w. and null for diminazeen at 7 mg/kg of b.w.
Target deployment led to the largest reduction in disease incidence from 28.1% in 2014 to 7.8% in 2017, an improvement in haematocrit from 24.27±4.9% to 27.5±4.6% and a reduction in calf mortality from 15.9±11% to 5.9%.

Conclusions
Improving the access to these interventions for different types of livestock and maintaining their effectiveness in the face of high tsetse challenges should be the primary focus of control strategies in areas of Togo.

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Background Tsetse-transmitted animal trypanosomosis is caused by a blood parasite, and from a symptomological point of view, it has a close a nity to malaria disease, whose impact on the e ciency of livestock production in sub-Saharan Africa is considerable [1]. The economic costs of this disease include direct and indirect effects and the costs associated with preventive measures implemented by farmers and the transmission control policies of governments [2], [3].
Togo is among the trypanosomosis endemic countries with the highest impact of this disease, and up to 90% of cattle and goat populations occur in substantially tsetse-infested villages [4]- [6]. Traditionally, the control of tsetse-transmitted animal diseases during livestock production in Togo is mainly based on local trypanotolerant cattle populations and the use of trypanocidal drugs [7], [8]. Before Togo's political crisis of the 1990s, a number of interventions against African animal trypanosomosis (AAT) were carried out to improve the areas with incidences of onchocerciasis that occurred over a long period of time based on the promotion of agriculture and livestock development [9]. Interventions have signi cantly reduced disease incidence in some areas [10]. Currently, although trypanosomosis is on the priority zoonosis list for West Africa, animal trypanosomosis in Togo is not listed as a priority for technical service intervention since the human form of this disease, named sleeping sickness, is declared eradicated (WHO, 2020). The rapid privatization of veterinary medicine in Togo has also led to a decline in animal health coverage and monitoring of cattle herds. Recent studies con rmed trypanocidal drug resistance in Togo [5], while the impact of this additional problem on the effectiveness of AAT control measures is poorly understood.
The Mô Plain is an area of the Guinean savanna in the central region of Togo with high agricultural potential over 1000 km 2 , adjacent to the Fazao-Malfackassa National Reserve [10]. This area is severely affected by the negative impact of AAT, despite its predominantly dwarf taurine-phenotype cattle herd [6], [10], [12]. In fact, the farmers in this locality improve herd productivity through practices characterized by various levels crossing between the local trypanotolerant dwarf somba populations and the large bovine breeds, in particular the zebu, which are trypanosensitive compared to taurine cattle.
Until 2013, the locality remained unconnected to outside areas, with long-term limited access to quality medicines, veterinary services, animal husbandry and new AAT control with pour-on technology [13]. This study was designed to assess the effect of α-cypermethrin pour-on application and diminazene aceturate (DA) for the treatment of trypanosome-related diseases on Mô traditional cattle herd health. The study was not designed to assess the effectiveness of interventions in relation to the absence of Tsetse y, so data were collected exclusively in the project area.

Study area
The study was carried out in Mô prefecture that has an area of approximately 1000 km 2 in the western part of the central region of Togo, bordering Ghana. The coordinates of the points of the survey are shown in Figure 1.
The prefecture has a subhumid climate typical of the Guinean zone with two main seasons: a dry season from November to March and a rainy season from April to October. The annual rainfall is in the range of 1074-1649 mm, and the number of annual rain days is 108-115 days [14]. The main vegetation is wooded Guinean savanna with surrounding gallery forests bordering the Mô River. The variation in rainfall greatly in uences livestock production. According to the information supplied by the technical staff of the project, the sedentary cattle population is estimated to be 40 cattle herds with 1437 heads [14]. Cattle in this area are the phenotypically taurine dwarf Somba (trypanotolerant) and their crosses with zebu that are maintained under a traditional extensive system (7). Cross-sectional analyses of several studies suggest animal trypanosomosis as a risk factor for local cattle production in this area [6], [10].

Study design
This study was implemented in the framework of the PDRI-Mô project. PDRI-Mô is an AAT control project using trypanocides and insecticides on cattle. This project, planned by the agricultural ministry, focused on all the sedentary cattle breeds in the area of the prefecture of Mô. To characterize the tsetse challenge across the prefecture, the grazing area, including the 40 cattle herds of the villages, was taken into account. The prefecture was divided into four strata of villages representing different administrative and cattle breeding areas: Bolohou, Djarkpanga, Tindjasse and Saiboude ( Figure  1). The strategy implemented for trypanocidal and α-cypermethrin pour-on treatment effectiveness involved cross-sectional (for baseline data collection) and longitudinal monitoring surveys. Thus, for the rst processing operation (March 2014), all 40 herds of the prefecture (counting an average of 2000 cattle) were treated. Following this, periodical (four month) treatment operations (trypanocidal and cypermethrin pour-on) were conducted from March 2014 to November 2017 according to a subjective assessment (report of the cattle owners and the technicians) of the level of challenge. The day before each intervention, per village, owners were encouraged to cooperate by preventing their cattle from going to pasture in the morning for free deworming and insecticide treatments against tsetse ies and ticks. The sick animals identi ed by owners were examined for AAT symptoms, and parasitological analysis of blood samples was conducted [16]. The monitoring consisted of recording calf mortality, tsetse abundance and new AAT cases, including their parasitological and anaemia status.

Entomological data collection
The collection of entomological baseline data is a prerequisite for the development of an appropriate control strategy [17], [18]. We conducted a cross-sectional monitoring survey of tsetse abundance in March 2014, March 2015, December 2016 and February 2017 using 20 biconical traps [19]. To estimate the abundance of the tsetse population, traps were deployed for 24 hours per day in each of the four villages. The selection of the trapping points considered accessibility (road) and hotspots of tsetse ies: small patches of remnant vegetation, gallery forests, cattle crossing points, etc.
Rapid trypanocidal drug sensitivity testing The DA and Isometamidium chloride (IMC) was used for treatment of infected cattle in the treatment group (Table 1). A comparative assessment of the sensitivity of the trypanosome parasite population to the two drugs was performed according to the rapid test [20]. This was a parallel study designed, blinded, randomized and negatively controlled. Ten random cattle herds (n = 120 cows) were selected based on the results of a parasitological survey performed in March 2016. The 120 experimental animals were all young males with an average age of 12 to 24 months. The detailed group design is presented in Table 1. The inclusion criteria were (i) phenotypic dwarf taurine cattle, (ii) trypanosome-related prevalence in herds of at least 10% and (iii) at least 12 trypanosome-positive cows in each sample of the herds. For each random herd, consent was obtained from the owner for inclusion in the study. Cattle tags with a unique identifying number were placed in the ear at the time of inclusion. The cattle received a randomized treatment (DA, IMC or distilled water as a placebo) using simple randomization that was concealed from the technician and the owner enrolling the animals in the test. The positive and negative control cattle per batch were added for the eventual natural clearing of parasites to provide trypanotolerance to the cattle population in the area. All cattle in each bioassay batch were sprayed with α-cypermethrin at a dose of 1 ml/10 kg of b.w. The aim of this insecticide treatment was to protect tested animals from new bites of trypanosome vectors, regardless of whether they became infected after drug treatment. Additionally, all the animals were treated with Ivermectin (1 ml/50 kg) one week before the drug treatment bioassay.
Post treatment (14 and 28 days) counts of the treated and control animals were used to evaluate the e cacy of the treatments [20]. Presence of trypanosomes in the Buffy coat smear was considered as indication of drug resistance. For all groups, the number of positive cows in each group was expressed as a percentage of the number of initial positive cattle before the treatment. At 14 days post treatment, for the cattle that were administered trypanocidal for apparent infestation, a second dose of DA was administered at 7 mg/kg of b.w., and the animals were monitored for two weeks thereafter.

Cattle mass treatment
Owners were encouraged to cooperate by identifying sick animals for free drug and insecticide treatments. The animals selected conventionally for treatment included any animal considered to present trypanosome-related symptoms or to be in poor condition [21]. Parasitological analysis of the blood samples [16] of the selected cattle was performed to con rm their health status before treatment. The packed cell volume (PCV) was read in a microhaematocrit tube after centrifugation for 5 min at 12 500 rpm [22]. Only positive and/or anaemic cattle (haematocrit or PCV lower than 25%) were administered trypanocidal and cypermethrin pour-on treatments [23]. DA was administered deeply intramuscularly at a dose of 7 mg/kg of body weight (b.w.). The dose of commonly used insecticide is 10 ml/100 kg of b.w. The treated adult cattle and all calves up to three months old were enrolled for deworming with ivermectin (1 ml/10 kg of body weight) [24], [25]. The purpose of deworming was not to systematically eliminate all internal parasites but to prevent anaemia.  (Table 2). A calf mortality mean rate of 15,9 ± 11% was recorded in 2014. This mean value was signi cantly higher than the baseline value of 5,9% in 2017 (P-value 0.001), showing a signi cant reduction in mortality ( Figure 5). Nevertheless, by village, there was a signi cant difference in the herd (village) at the beginning and end of the follow-up.

Discussion
This study provided a unique opportunity to quantify the attributable effect of trypanosome and tsetse control efforts on animal trypanosomiasis in high transmission settings of the Guinea savanna in Mô. Our data are important within the context of AAT control planning locally in endemic areas with crossbred trypanotolerant-trypanosensitive cattle breeding and important elsewhere on ranches where AATs a similar epidemiologic pattern.
Our results con rm previous observations that salivary AAT vectors in this area are G.t and G.pp, G.ms, G.f [6] and G.l [10]. The baseline entomological survey in the present study was conducted by collecting data and monitoring at tsetse-cattle herd contact points. Therefore, the tsetse TAD evaluated in the Mô area is likely to be higher than that reported in this study. In contrast, species diversity and TAD values were higher than those previously reported in the same area [6]. This implies speci c concentration points and a temporary rise in forest tsetse species from the wildlife in the Mazao-Malfakassa forest including in Mô area.
Tsetse density as a whole decreased signi cantly; however, it remained relatively high, at slightly over the maximum of 0.26 measured in the north of Togo [10]. Several studies have shown that the insecticide pour-on control strategy has a limited effect on tsetse y density. A survey carried out as part of pilot projects with the selected insecticide method showed a signi cant impact on tsetse y density, with a reduction of 99,55% [27]. Two case factors accounted for these lapses, apart from a small tsetse density, which was between 0.2 and 0.7, the frequency of treatment processing operations (every two months) and the high insecticide-treated cattle coverage rate, which was 100%. In the agropastoral area of Yale in Burkina Faso, [24] obtained a good result of a 90% reduction of Glossina density. The technique combined the use of insecticide (deltamethrin 1%) in pour-on and insecticide-impregnated screens against tsetse (the frequency of treatment processing operation was twice a month). The relative low e cacy recorded the present study can be partly explained by the absence of intervention in the third treatment, which was meant to ensure health coverage from November 2016 to February 2017. In addition, in the present study, treatments were limited to three times a year, and for each intervention, only 10-20% of the cattle were treated in a herd. This time frame of up to 4 months between two consecutive operations provides a su cient window of time for the rise of the new tsetse population from buried pupae [28], [29].
The main categories of local cattle breeds in Togo are dwarf trypanotolerant cattle and their crossbreeds (dwarf x zebu) [7]. The use of insecticide cattle treatments in such heavily tsetse-infested areas has not been shown to be particularly effective against tsetse ies [23]. However, it is possible to prevent other insect bites, such as ticks, that can cause illness and thus contribute to the poor health of the animals. In the present study, although insecticide application was carried out on tick-infested cattle, we did not evaluate the impact of this operation. In addition, limiting insecticidal interventions makes it possible to maintain minimum contact between cattle and tsetse ies, which is essential to allow multiplication of parasites prior to drug treatment to induce immunity in the host [30].
The high incidence of AAT disease in Mô is in agreement with previous observations [6]. With such a high incidence, previous routines and substantial use of trypanocidal drugs by farmers can lead to the development of trypanosome resistance (T.v and T.c are the two strains identi ed in the area) to the only available DA/IMC drugs. However, in the present study, no baseline data were established. This testing of operation effectiveness by monitoring staff aimed to validate the approach and previous ndings concerning trypanocidal drug resistance elsewhere in West Africa [31].
Evidence of a relapse of parasites, i.e., presence of trypanosomes in the blood after trypanocide treatment, can occur due to the appearance of trypanosome populations from other sites, such as cerebrospinal uid [32]. This might have also been observed due to new vector contamination in the eld when treatments failed to protect animals against bites from tsetse ies after certain days. Given the cattle production system in this locality, a failure of the immune system to control the multiplication of chemically resistant parasites in trypanotolerant cattle may lead to a higher impact of trypanosomosis in this rearing system.
The mean PCV after three years of treatment was 1.4 absolute % higher than the value recorded by Talaki et al. (2014) and 3.2 absolute % higher than the initial value before processing operations. Consequently, as an overall health impact of control, the incidence of the disease decreased to 7.4% in 2017 (reduced by 20.3 absolute %) but remained almost stable and higher than the value of prevalence (2%) recorded by [27] in the North of the area in the dry savanna. There is no evidence to date to indicate that AAT transmission can be interrupted with existing tools in the study area of high tsetse challenge. Some important insights into the feasibility of reducing and maintaining tsetse TADs at a low level in areas of high transmission were obtained on the Adamawa Plateau, Cameroon [33].
The average mortality rate in 2017 was slightly above average at 3.37% in somba herds (north of Togo) based on Ibrahim et al. [34] but lower than 15.9 ± 11% in 2014 and the average (mortality rate of 10.98 ± 3.41% for crossbreed and 11.85 ± 4.19% for dwarf taurine) between Mô and Sokode recorded by Soudah et al. [12]. This result can partly be explained by interference with other diseases while not ignoring a high vulnerability to the reinvasion of tsetse from the Fazao-Malfakassa wildlife forest reserve in Mô area.
This situation suggests that taking into account all health conditions can lead to more effective results in the herds.

Conclusions
The AAT processing operation in the Mô area was not su cient to avoid trypanosome parasite transmission in Mô prefecture, as tsetse density was still relatively high. The present study was conducted in a highly endemic trypanosomosis area where trypanocides and insecticide-treated cattle were introduced for the rst time. The low haematocrit levels initially recorded clearly re ected the severity of the disease in the animals. The impact of animal health control has thus resulted in a reduction in anaemia and the mortality of calves. As a result, we recommend continuing this activity and extending it to other production areas targeted by the "International Fund for Agricultural Development: IFAD-Livestock Togo" by integrating other technological packages to improve herd productivity. Any data generated in this study is included in the published article.
Competing interests "The authors declare that they have no competing interests"

Funding
The corresponding author received Ph.D research grants from WAAP-Togo (IDA N°5955-TG) for eld data collection and personal contribution from authors for language editing service. Change in the incidence of AAT in the control villages Yearly mortality occurring of calves during the trypanocide and insecticide intervention

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