A total of 37 districts were sampled in all 5 regions of Uganda ( Figure 1 ). The central region, which combines the northern and southern Buganda sub regions, was represented by 11 districts (Sembabule, Gomba, Masaka, Kyotera, Lyantonde, Nakaseke, Nakasongola, Lwero, Mukono, Kayunga, and Rakai), with a total of 106 cattle farms sampled. The Eastern region, which combines the Busoga, Sebei, Bugisu and Teso sub regions, was represented by 6 districts, which included Jinja, Iganga, Mbale, Sironko, Kapchorwa and Kumi. A total of 59 farms were sampled from the Eastern region. The Mbarara, Isingiro, Rukungiri, Sheema, and Buhweju districts represented the southwestern region, which comprises the Ankole and Kigezi sub regions, with a total of 50 farms sampled. Similarly, the Kasese, Ntoroko, Kabarole, Kamwenge, Kiryandongo, Masindi, Buliisa, Kyankwanzi, and Hoima districts were sampled from the western region (comprising the Tooro, Bunyoro and Rwenzori sub regions), with a total of 89 farms sampled. The northern region, which combines the Acholi, Lango, Karamoja and West Nile sub regions, was represented by the Moroto, Kotido, Adjumani, Gulu, Lira and Arua districts.

In each of the selected districts, the two sub counties with the highest number of cattle were identified with the help of the district veterinary officer. From each of the sub counties, one parish with the highest number of cattle was identified with the help of the subcounty extension officer. A list of cattle farms in each of the selected parishes was generated by the extension officer, and five cattle farms were selected randomly from the pool. The selected farms were informed, and a request to visit their farms was made by the extension officer. Those who refused to participate were replaced by other randomly selected farms. Appointments were made with the farm owner to visit the farm to collect ticks and relevant data (GPS coordinates and farm address).

Upon arrival at the farm, a short meeting took place between the research team, extension officer and farmer to explain the purpose of the study and to obtain informed consent from the farmer to participate in the study. The administration of the semi-structured questionnaire for assessing the acaricides used for tick control was conducted for each farm visited. The GPS coordinates for each of the selected farms were collected and recorded among the farm details. Randomly selected cattle were mobilized and restrained using cattle crushes, and in farms where cattle crushes were absent, ropes were used to restrain the cattle for tick collection across the various predilection sites (ears, udder, dewlap, head, neck, brisket abdominal region, tail, fore and hind legs) for species identification and chemical tick assays.

Tick sorting and taxonomy were conducted with the aid of a stereomicroscope following the taxonomical [18] at the Research Center for Tropical Diseases and Vector Control (RTC), College of Veterinary Medicine, Animal Resources & Biosecurity, Makerere University. Engorged female ticks of the same species from the same farm were selected and incubated in groups of 3 - 4 ticks per culture tube and labeled. The incubation tubes containing the ticks were transferred to an incubator set at 27˚C ± 1˚C and 80% relative humidity. The incubation process was monitored daily to ensure that the conditions remained constant and to check for possible egg laying and egg hatching. The larvae were left to mature for 10 - 14 days after hatching, and would be ready for acaricide bioassays.

To determine the susceptibility of the tick larvae to acaricides, we adopted the FAO’s recommended larval packet test [19] and was performed from the Research Center for Tropical Diseases and Vector Control (RTC).

Commercial formulations of commonly used classes of acaricide in Uganda were selected. These included Synthetic pyrethroids (deltamethrin), amidines (amitraz) and co-formulations (combination of chlorpyrifos and cypermethrin). The use of commercial formulations is recommended for large scale acaricide resistance surveillance programs (countrywide) involving over 200 tick populations. Also, use of technical grade (chemical reference standards) is not economically feasible in a resource constrained country due to associated high costs of obtaining the reference chemicals. To address issues regarding the quality of commercial formulations, we sourced the products directly from the importer (local technical representative of the manufacturer) whose products undergo regular quality assessment by the regulator for compliance. For internal quality assurance, susceptible populations of larvae of Amblyomma variegatum were exposed to the recommended concentration of each of the commercial acaricide formulations. Only those that achieve the 100% mortality within 24 hours are used in the experiments.

A mixture of trichloroethylene (TCE) and analytical grade olive oil was prepared in a ratio of 2:1 respectively. The solvent was prepared.

The manufacturer’s recommended concentration (discriminating concentration (DC)) was considered the diagnostic dose for all the chemicals tested. The FAO recommended larval packet test (LPT) was adopted for the acaricide susceptibility tests. The recommended (manufacturer) concentrations of deltamethrin, amitraz and the co-formulated acaricides (chlorprifos + cypermethrin) used in this study were 0.05 mg/ml, 0.25 mg/ml, and 0.5:0.05 mg/ml (chlorpyrifos: cypermethrin), respectively. Each tick population was exposed to the recommended concentrations of all three classes of acaricides.

Filter paper (Whatman No. 1, Whatman, Madstone, United Kingdom) was used as a substrate for deltamethrin, and a combination of chlorpyriphos and cypermethrin and Nylon fabric was used for amitraz. The LPT was conducted following methods previously published by several scholars [15] .

The LPT results were assessed after 24 hours of incubation of the packets. The numbers of living and dead ticks were counted and recorded for each chemical, each tick species and each replicate. Mortalities were expressed as percentages of the total number of larvae exposed to the acaricide. The data from the laboratory tests and the Global Positioning System (GPS) coordinates for the sampled farms were transferred into Microsoft Excel software, where percentage mortalities were calculated. From the percentage mortality results, a given tick population was categorized as susceptible if the percentage mortality value was ≥ 90% or resistant if the percentage mortality was < 90%. Resistant tick populations were further categorized as single resistance or multiple acaricide resistance. A tick population resistant to only one class of acaricide was categorized as single acaricide resistance whereas those that were resistant to at least two acaricide classes were categorized as multiple acaricide resistance.

The acaricide resistance maps were generated on the basis of tick species and acaricide classes across all regions of Uganda using ArcGIS® software for desktops version 10.4.1. The GPS data (coordinates) were plotted in ArcGIS® software to generate the locations of the farms. Statistics for each district were generated via geostatistical techniques in ArcGIS® software. The results were presented at the district level and were visualized via charts and graduating colors. The red and maroon colors represent resistance, whereas the green color represents susceptibility.

Informed consent was obtained from farm owners or their representatives as evidence of voluntary participation in the research. The researchers upheld the principles of confidentiality and anonymity. The cattle were handled humanely at all stages of tick collection to ensure that welfare was guaranteed.

Out of the 357 farms sampled in all the regions of Uganda, 37.5% (134), 32.5% (116), 20.4% (73), 2.0% (7) and 1.7% (6) were using acaricides in the classes of co-formulations, amidines, synthetic pyrethriods, organophosphate and crop agrochemicals for tick control on their farms respectively ( Figure 2 ). Interestingly, 6% (21) of the farms were not using any of the chemicals for tick control.

A total of 201 tick populations were tested using standard LPT test. Generally, acaricide resistance was detected for all the major acaricide classes commonly used for tick control on farms in Uganda ( Table 1 ). The highest percentage (80.1%) of tick populations tested in the country were resistant to deltamethrin (a synthetic pyrethroid). Contrary, the 201 tick populations tested exhibited lower resistance against amitraz (57.7%) and co-formulated acaricides containing cypermethrin plus chlorpyrifos (52.2%). In terms of susceptibility, 47.8% of the tick populations tested were susceptible to co-formulated acaricides containing cypermethrin plus chlorpyrifos.

At the regional level, a total of 44 populations of R. decoloratus ticks were tested from the southwestern region. They were highly (100%) resistant to deltamethrins and amitrazs (88.6%). On the contrary, majority (79.5%) of the R. decoloratus tick populations tested from the southwestern region were susceptible to co-formulated acaricides containing cypermethrin and chlorpyrifos. Similarly, R. appendiculatus tick populations (n = 14) from the southwest were also highly resistant to deltamethrin (100%) but largely susceptible to amitraz (92.9%).

The R. decoloratus tick populations (n = 25) from the Central Region were highly resistant to all three classes of acaricides commonly used in Uganda, with 100%, 96% and 96% of the populations tested showing resistance to deltamethrins, coformulated acaricides (cypermethrin and chlorpyrifos) and amitrazs, respectively. The results for R. appendiculatus tick populations were not different from those observed for R. decoloratus tick populations for deltamethrins and co-formulated acaricides (cypermethrin and chlorpyrifos). However, R. appendiculatus tick populations (n = 44) were largely (75%) susceptible to amitrazs.

The R. decoloratus tick populations (n = 18) from the western region were highly resistant to both deltamethrins (100%) and amitrazs (94.4%). Only half of the tested populations of R. decoloratus ticks from the western region were susceptible to co-formulated acaricides (cypermethrin and chlorpyrifos). Moreover, R. appendiculatus tick populations (n = 25) from the same region presented quite different results, with a simple majority (of the tested populations) being susceptible to deltamethrins (64%) and coformulated acaricides (56%). However, 72% of the same tick species population was resistant to amitrazs ( Table 1 ).

Although a total of four tick species were tested from Eastern region, the majority of the tick populations tested were R. decoloratus (n = 9), which exhibited high (89%) resistance to deltamethrins, followed by both amitrazs and coformulated acaricides at 55.5%. We detected acaricide resistance to deltamethrins among the Hyalomma spp. population tested. Similarly, we detected amitraz resistance among the R. evertsi evertsi tick population tested from the Eastern Region. The R. appendiculatus tick populations (n = 2) from the Eastern Region were resistant to deltamethrins and co-formualated acaricides. The tick populations from the northern region, including R. evertsi evertsi, Hyalomma spp. and R. appendiculatus, were highly (100%) susceptible to all three major acaricide classes commonly used in Uganda ( Table 1 ).

Keywords: R = Resistant, S = Susceptible and N = Tick populations tested.

The findings revealed that the multiple acaricide-resistant R. appendiculatus tick populations were widely distributed in the majority of the districts located in the Central, Western and Southwestern regions of Uganda. However, acaricide susceptible R. appendiculatus tick populations were detected in Kasese and Ntoroko districts (Western region) and in the districts of Sheema, Kazo and Kiruhura (South-western region). The R. appendiculatus ticks from the Eastern and Northern regions of Uganda were largely susceptible to all the major acaricide classes commonly used in Uganda ( Figure 3 ).

The multiple acaricide-resistant R. decoloratus tick populations were confirmed to be widely distributed within the southwestern, western and central regions of Uganda. Within the southwestern and western regions, acaricide susceptible R. decoloratus tick populations were only detected in some farms located in Rakai and Buliisa districts respectively. On the other hand, the eastern and northern regions were still largely free of acaricide-resistant R. decoloratus tick populations. The ticks collected from the northern region were highly susceptible to all the major acaricide classes commonly used in Uganda ( Figure 4 ). However, multi

acaricide resistant R. decoloratus tick populations were prevalent on farms located in Mbale and Jinja districts (eastern region).

Resistance to deltamethrins across major tick species was confirmed to be widely distributed across major cattle keeping regions of Uganda, such as the southwestern, western, central and eastern regions of Uganda. Tick populations susceptible to synthetic pyrethroids were only detected on some farms in Masindi district (western region). However, the northern region of Uganda (Karamoja, Lango, Acholi and West Nile) was free of pyrethroid-resistant ticks at the time of the study ( Figure 5 ).

Amitraz/amitraz resistance was widely distributed across major tick species and across the entire western, southwestern and central regions of Uganda. Tick populations susceptible to amitraz were only detected on some farms located in Nakasongola district (central region). Isolated cases of amitraz resistance were also confirmed to be sporadically distributed in some study districts (Mbale and Kumi districts) within the Eastern Region. At the time of the study, amitraz-resistant ticks were not detected from the entire northern region of Uganda ( Figure 6 ).

Resistance to combination of chlorpyrifos and cypermethrin was widely distributed among districts located in the central, southwestern and western regions of Uganda. However, a combination of chlorpyrifos and cypermethrin had a fairly good effect on ticks collected from some isolated districts within the hard-hit regions. These districts include Sheema (southwestern), Kamwenge, Buliisa and Ntoroko (western) and Nakasongola (central). Resistance against combinations of chlorpyrifos and cypermethrin across tick species was also confirmed to be widely distributed within some districts located in the Eastern Region, such as Jinja, Iganga and Mbale. The rest of the Eastern region study districts, together with all the study districts from the northern region were free of tick resistance against combinations of chlorpyrifos and cypermethrin at the time of the study ( Figure 7 ).

In general, tick acaricide resistance to at least one acaricide molecule was confirmed to be widespread within the western (Tooro, Bunyoro and Rwenzori subregions), southwestern (Ankole and Kigezi sub regions) and central regions (North and Southern Buganda sub regions) of Uganda. Some pockets of tick resistance were also confirmed within the Eastern region in the districts of Mbale, Jinja, Iganga, Sironko and Kumi. Acaricide susceptible tick populations were only detected on some farms in Masindi district (western region). However, the northern (Lango, Acholi and West Nile sub regions) and eastern regions (Busoga, Bugisu, Sebei, Teso, and Karamoja sub regions) of Uganda were largely free of tick acaricide challenges ( Figure 8 ).