Study area
The study was carried out in Mô prefecture that has an area of approximately 1000 km2 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 influences 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 first 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 flies and ticks. The sick animals identified 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 flies: 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).
Table 1: Composition of the experimental batches for the treatments
Herd
|
Batch
|
Treatment
|
12 animal/experimental herd
|
10 Positive
|
40 young male treated with diminazen
|
40 young male treated with isometamidium
|
20* young male treated with distilled water
|
2 Negative
|
20* young male treated with distilled water
|
*: Animal was used as control.
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 efficacy 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 confirm 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.
Data analysis
Tsetse fly apparent densities were evaluated as the number of tsetse flies per trap per day (TAD), calculated by dividing the total number of tsetse flies captured by the product of the number of functioning traps used to catch them and the number of days for which the traps were operational [26]. By herd, mean annual birth-to-weaning calf mortality was determined by dividing the number of deaths by the number of alive births. For each processing operation period, the numbers of new sick cattle were expressed as a percentage of the anterior hull cattle count in the 40 herds. The data was recorded into a Microsoft excel spread sheet to create a database and transferred to the statistical package for social sciences (SPSS) version 20 software programs of the computer for the statistical analysis. The Chi-square test was used to compare the frequency of trypanosome related species and frequency of the disease cases with respect to absence or presence of infection) at a precision level of 5%. Student-Newman-Keuls was used to compare between year mean calf mortality and mean TAD. ANOVA multiple comparisons test was applied by comparing the PCV values with respect to year and absence/presence of infection. The trypanosome population of the study area was considered to be phenotypically resistant to the given trypanocidal drug dose when relapse occurred in more than 20% of the batch [20].