Amongst other tick species in the world, R. decoloratus is the most widely distributed tick species and considered as the most important external parasite to livestock, particularly in cattle farming [1]. The R. decoloratus is known as the indigenous tick to the Africa continent and widely distributed in both tropical and subtropical regions. Rhipicephalus decoloratus is regarded as the major vector for transmission of tick-borne diseases (TBDs) such as Babesia bigemina, Anaplasma marginale and A. centrale to cattle, unlike its counteracting specie R. microplus which can also transmit B. bovis amongst other pathogens [2,3]. Heavily infestation of ticks results in great economic losses on cattle production through the reduction of milk yield in cows, meat and damages the skin. However, over time, the R. decoloratus ticks is known to be the resistant tick to almost every application of registered acaricide chemical, thus increases its rapidity spread into non-endemic areas [4]. Tick's lifecycle and worldwide distribution of the tick infestation as well and indiscriminate acaricide use by cattle farmers have proved to be the most contributing factors to the rapid establishment of the tick resistance to acaricides compounds [5]. In South Africa, there are three synthetic acaricides chemicals groups used to control ticks; organophosphates, amidines and pyrethroids [6,7]. Of these acaricides groups, the pyrethroids and organophosphates are the most commonly used insecticides groups by most resource-limited farmers [8,9]. However, over the past fifty years, sodium arsenite was the first successful acaricide to be used in South Africa until the first report of the resistance detection on R. decoloratus [10]. Thereafter, Benzene hexachloride (BHC) took over to the arsenic as an alternative in the Dichloro-diphenyltrichloro-ethane (DDT) form [10]. Since then the incidence of arsenic-BHC-resistant blue ticks was reported rapidly increasing along the coastal regions of the country [11]. In addition to this, [7] reported the first record of R. decoloratus being resistant to organophosphate in the eastern regions of the ECP and no reports yet on the northern parts of the province. To that there are several bioassays to be used in the evaluation of the tick susceptibility to acaricide chemicals includes adult immersion test, larval packed test [12] and larval immersion test [13]. Recently a new bioassay method, the larval tarsal test was developed in comparison to the larval packet test and both tests were successfully able to detect resistance to organophosphate, amitraz and coumaphos [14]. The larval tarsal test was proved to be more advantageous than the larval packet test since it allows a large volume of doses and compounds in a short period of time and less number of the engorged females [15]. Status of resistance for tick population is normally established through ticks exposure to the unique type of dosage guided by the information of a susceptible reference strain, thereafter, the discriminating dose survival indicates acaricide resistance [16]. Moreover, [15] articulated that, several acaricede doses can be used for ticks in order to develop acaricide resistance for up to 50 % or 90 % mortality. Thus, to this phenomenon, the objective of the current study was to determine acaricide resistance of R. decoloratus larvae collected from cattle herds of the north-eastern region of the ECP using larval immersion test and to access their susceptibility to three acaricide groups (organophosphates, Amidines and Synthetic pyrethroid) commonly used by the cattle farmers.

Ethical Consideration

Before the commencement of data collection, ethical clearance was obtained from the Research Ethics Committee at the University of Fort Hare (Reference number; MUC021SYAW01). All experimental procedures were conducted as per the moral standards of experimentation given by the committee of ethics on animal use of the Society for the Prevention of Cruelty to Animals (SPCA) [17].

Study Site

This study was conducted in thirty-three (33) communities in Elundini, Senqu and Walter Sisulu municipalities under Joe Gqabi District in the ECP. Elundini municipality is situated at 1600 m above sea level with the average annual rainfall ranges between 800 to 1200 mm and the average annual minimum temperature of 13 °C and maximum temperature of 22 °C under Southern Drakensberg Grassland [18]. Senqu municipality is situated at 1000 m and 1500 m above sea level with the annual average temperature of -16 °C during winter and 42 °C during the summer season. The annual rainfall ranges between 1000 mm and 1400 mm under Montana Shrubland [18]. Walter Sisulu municipality is situated at the altitude of 1000 m and 1500 m above the sea level. The annual average temperature ranges between 15 °C during winter and 30 °C during the summer season under the Mixed Nama Karoo vegetation type and its annual rainfall ranges between 1000 to 1400 mm [18].

Experimental Animals

Random selections of ten animals were selected from the cattle herds for ticks sampling at each dip tank during dipping days from November 2018 to September 2019. At all times all the selected animals were older than 12 months, included both sexes. The cattle in the study areas were composed dipping was done fortnightly during summer and once in a month time during the winter season. Ticks sampling from cattle was done before dipping to avoid a bias of tick population. In these localities, water supply to the dip tank was reported to be the major challenge which limits farmers to use plunge dipping system during the dry season. Cattle largely depend on natural pastures for feeding with a continuous grazing system.

Ticks Collection and Transportation for Acaricide Testing

Regardless of the type of test to be used for acaricide resistance, engorged female ticks were collected from grazing cattle between 08h00 and 09h00, this was done because most of the engorged ticks drop off the host in the early morning as described in Chapter 4, Section 4.2.4. A sample of 20–30 adult engorged female R. decoloratus ticks were collected from at least 10 cattle at each dip tank and placed into plastic collection bottles containing absorbent paper and with perforated lids at constant room temperature of ±28°C and >70 % relative humidity. Each collection bottle was labelled with information on the date of collection, farm name and number of cattle sampled. At all times after collection, ticks were immediately dispatched to the Acaricide Resistance Testing Laboratory in the Department of Zoology and Entomology, University of the Free State for acaricide resistance testing. Upon arrival at the laboratory, engorged female ticks were washed on a sieve using clean tap water and all damaged and undersized ticks (below 150 to 350 mg) were discarded. Ticks were then air-dried in absorbent paper, placed in a glass flask and incubated [6-7]. Ticks were daily monitored and checked until oviposition commenced. After the first egg production of about approximately +35 days then ticks were further monitored daily for hatch date establishment, which was known to be the day where hatched larvae were approximately 70 %, thereafter the acaricide resistance testing was performed on larvae between the ages of 15-21 days [19-20].

Acaricide Chemicals Used In the Study

The three dip formulations were used for acaricide resistance testing in the study; (i) Organophosphate (Supadip), (ii) Synthetic Synthetic pyrethroids (Pro-dip) and (iii) Amidines (Tritex). These compounds were chosen because they are regularly used in South 

Africa as well and commercially available and all registered according to Act 36 of 1947 for tick control. The concentrations of the acaricide compounds in which tick larvae were exposed into the SLT include the standard recommended field concentrations for each acaricide as prepared from 1 % stock solution diluted from each acaricide group as shown in (Table 1). The used concentrations consisted of Cypermethrin 0.015 and 0.03ppm, Chlorfenvinnphos 0.03 and 0.05ppm and Amitraz 0.025ppm and double-distilled water was used to prepare these concentrations and one was used as the control. For each concentration test and distilled water, 10 ml were placed into the labelled test tubes that were used for the next step of acaricide testing. At all times, thorough mixing of the concentration and water was done during the preparation process to ensure uniformity of the acaricide solution.

The acaricide groups used are represented as follows:

• Organophosphates: Chlorfenvinphos 30 % m/v (“Coopers Supadip®” - Fort Dodge, Bayer Animal Health Pty Ltd.), registration number G1284 (Act 36/1947), South Africa.
• Amidines: Amitraz 12.5 % m/v (“Triatix®” - Intervet South Africa Pty. Ltd.), registration number G845 (Act 36/1947), South Africa.
• Synthetic Pyrethroids: Cypermethrin 20 % m/v (“Pro-Dip®” - Fort Dodge, Bayer Animal Health Pty Ltd.), registration number G2311 (Act 36/1947), South Africa (Table 1).

Table 1: Acaricide resistance test dilutions.

Shaw Larvae Immersion Test

Upon arrival at the laboratory, engorged female ticks were handled according to the prescribed Standard Operating Procedure (SOP) [13]. In short, SOP entails that engorged females were washed on a sieve using clean tap water and all damaged, not engorged and those already started to lay eggs were discarded. Ticks larvae were exposed to the field concentration for acaricide resistance test using SLIT and efficacy was determined by the percentage of larvae killed through exposure. An above 80 % mortality percentage was considered to be effective and a percentage below 80 % but above 50 % was considered as an indicator for resistance development, while below 50 % mortality was considered as resistant.

Larvae Exposure to the Acaricide

The conical flask containing larvae samples for testing was placed into the water and the petri dish. A 24cm diameter round filter paper was then placed in the stainless-steel tray for soaking up any form of water droplets that may be spill during the commencement of the actual test. A foil plate that consisted of two circular filter papers of about 11cm diameters each was placed into the 24cm filter paper. The resistant test begins when the cotton stopper was removed from the flask using forceps and placed into the side of the 11cm filter papers in the pie plate. Remaining larvae in the neck of the flask were then removed using a demarcated control brush and brushed into one filter paper and coved. The flask was then closed using the cotton stopper and the test tubes containing the control were vortexed for 10 seconds before being poured over the filter paper sandwich. When liquid poured into the filter paper timer was started and the same process was repeated for 60 seconds and the uncontaminated bush was used to transfer tick larvae into the filter paper. After all concentrations were tested, masking tape was then used to remove all larvae that escaped into the cotton stopper and the flask was placed back into the incubator box and moved to the incubator 

room.

Larvae Post-Exposure from the Acaricide

Starting from the control exactly 10 minutes later after larvae exposure, the sandwich filter paper was removed from the plate using forceps and some water was drained off into one corner of the 24cm filter paper, thereafter, the foil plate was discarded. The sandwich filter paper was then pulled apart and placed in the dry parts of the 24cm filter paper to allow excess liquid to be drawn. Once more designated brush was used to move larvae into the pre-labelled filter paper envelope and masking tape was used to prevent escaping larvae from the filter paper. Two envelopes containing chemicals were then tightly clipped together and placed into the incubator for 72 hours at ±28°C and >70% relative humidity. This process was then repeated in each of the field concentrations and tested using a new foil plate and 24cm paper. Acetone was used to clean the trays between the use of each chemical concentration used and separate incubator boxes were used to separate the chemical and control envelopes in the incubator room.

Larvae Mortality Counting

The filter paper envelopes were removed from the incubator container after 72 hours and all live and dead larvae were counted. Masking tape was removed from the edges of the control envelopes and was left opened and live larvae were then counted on a 24cm filter paper and attention was paid to avoid larvae that may be running away during counting. The total number of live larvae was then documented in the corner of the envelope. All the dead larvae were poured and counted into the 24cm filter paper below. Therefore, the mortality percentage was determined by counting the number of live and dead larvae. Corrected mortalities were determined using water control results which they had less than 10 % with a use of [21] formula as follows;

CM% = (% i-%c) / (100-%c) × 100/1

Where;

 CM % = corrected mortality

 % i = % mortality in concentration

 % c = % mortality in water control

The SLIT was only performed on the R. decoloratus larvae as engorged R. microplus ticks could not meet the required sample size for resistance testing. Each resistance range was presented by its specific colour from Red= Indications of Resistant, Yellow= Developing Resistance, Blue= Effective Reservation and Pink= Susceptible/Effective as shown in (Table 2) below.

Table 2: The range of resistance percentages used to display the larvae resistance.

Resistance Profile of Amidine, Organophosphate and Synthetic Pyrethroids

(Table 3) below shows the defined resistance development of R. decoloratus larvae exposed at different field concentration levels of amidine, organophosphate and synthetic pyrethroids. Amidine at concentration of 250ppm presented the largest proportion which was described as susceptible (49 %) to the used chemical, with 30 % developing resistance and 21 % described as effective reservation and there was no resistance recorded in this chemical concentration. Organophosphate at concentration 300ppm displayed the greatest proportion of effective reservation (45 %) with 33 % were susceptible to the chemical, 18 % developing resistance and only 4 % were considered to be resistant. On the other hand, fairly set of results were observed when larvae exposed to organophosphate at concentration 500ppm where 47 % were described to be susceptible to the used chemical treatment, 32 % showed effective reservation and 21 % indicated as developing resistance and was interesting to note no resistance on tested samples in this concentration level in this chemical. The synthetic pyrethroids results at concentration 150ppm, presented 44 % samples tested in the chemical to be susceptible to the treatment, with 37 % effective reservation, 12 % developing resistance and 7 % conceded to be resistant. However, on the other hand, synthetic pyrethroids at concentration 300ppm recorded the greatest proportion of effective reservation (34 %), followed by developing resistance (30 %), with 23 % samples displayed as susceptible and 13 % sustain the resistance.

Table 3: Resistance status of amidine, organophosphate and synthetic pyrethroids used at 150ppm, 250ppm, 300ppm and 500ppm field concentration levels.

Rhiphicephalus Decoloratus Larvae Resistance Profiles Exposed To Amidine

Figure 1 shows the mortality counts of R. decoloratus tick’s larvae collected from Elundini, Senqu and Walter Sisulu exposed to amidine filed concentration. Elundini, Walter Sisulu and Senqu described being susceptible towards exposure to amidine chemical with the mortality counts of 50 %, 47 % and 44 %, respectively. More so, mortality counts for effective reservation were largely observed in Walter Sisulu (40 %), 38 % at Senqu and lowest mortality counts at Elundini region 35 %. Resistance development of the larvae to the chemical was greatly observed at Senqu (18 %), 15 % at Elundini and 13 % at Walter Sisulu region and ticks did not show resistance when exposed to this chemical across the localities (Figure 1).

Figure 1: Resistance profiles of R. decoloratus larvae exposed to amidine.

Rhiphicephalus Decoloratus Larvae Resistance Profiles Exposed To Organophosphate

(Figure 2) shows mortality counts of R. decoloratus tick’s larvae collected from Elundini, Senqu and Walter Sisulu exposed to organophosphate field concentration. Susceptible mortality counts were largely observed at Senqu (52 %), 48 % at Elundini and lowest Walter Sisulu had the lowest mortality counts 42 %. Whereas the highest mortality counts for effective reservation were recorded at Senqu 42 %, Walter Sisulu with 37 % and 31 % for Elundini. The highest larvae within the resistance development range to the chemical were greatly observed at Walter Sisulu (19 %) followed by Elundini (15 %) and lowest counts (6 %) at Senqu region and 6 % and 2 % mortality counts were observed at Elundini and Walter Sisulu regions, respectively. There was no tick resistance in Senqu region at organophosphate exposure. 

Figure 2: Resistance profiles of R. decoloratus larvae exposed to organophosphate.

Rhiphicephalus Decoloratus Larvae Resistance Profiles Exposed To Synthetic Pyrethroids

(Figure 3) shows mortality counts of R. decoloratus tick’s larvae collected from Elundini, Senqu and Walter Sisulu exposed to synthetic pyrethroids concentration. Almost half of the larvae mortality counts exposed to the chemical across the localities were described to be susceptible with 50 % at Senqu, Elundini (48 %) and Walter Sisulu (47 %), respectively. Similarly, larvae mortality counts on effective reservation category were 40 % at Senqu, 39 % at Walter Sisulu and lowest counts at Elundini (26 %). The highest larvae within the resistance development range to the chemical were observed at Elundini (17 %), 9 % Walter Sisulu and 7 % Senqu. On the other hand, the greatest mortality counts larvae described to be resistant to the applied chemical were largely observed at Elundini (9 %), 5 % at Walter Sisulu and only Senqu had the lowest counts (3 %) compare the other localities (Figure 3).

Figure 3: Resistance profiles of R. decoloratus larvae exposed to synthetic pyrethroids.

In South Africa, several study [22-27,15], have documented studies of the R. microplus resistance to most commonly commercial used acaricides chemical for ticks control. Moreover, these studies also reported the displacement of the African blue tick (R. decoloratus) by the Asian cattle blue tick (R. microplus), however, there was no displacement in this study. This study found patchy engorged R. microplus tick specimens in each study site during tick collection days in which its samples were not enough for resistence testing. Similar findings were also reported by [28] who reported fewer R. microplus counts in the study that was conducted in the coastal regions of the ECP in which the collected R. microplus samples did not meet the sample size for resistance testing. These findings were attributed to that, R. microplus might be possible susceptibly to the currently used acaricides in the localities. It is further articulated that, the resistance information of the R. decoloratus in South Africa, in particular in the ECP is mostly outdated as more focus has been shifted towards the invasive tick species, R. microplus, as a result resistance profile information need to be updated [6,7] . Over the past 10 years the three acaricide compounds have been used to control ticks as they are known by their low toxicity to cattle and to other animal species in which ticks are controlled. Briefly, these chemical acts as an octopamine receptor when applied into the tick leading to decrease neuron numbers in which are usually active and subsequently resulting in paralysis and death [29]. The mechanism of tick resistance to acaricide is described by an increase of metabolic activity of the tick that produces enzymes for the metabolism which detoxify any toxic substance as soon as possible before affects the target sites [30]. The current study found that majority of the tick were considered to be susceptible at exposure to different acaricide field concentrations of amidines (49 % at 250ppm), organophosphates (33 % and 47 % at 300ppm and 500ppm, respectively) and synthetic pyrethroids (44 % and 23 % at 150ppm and 300ppm, respectively). The reason for this might be influenced by low tick counts in the study areas, in particular the blue tick than expected. The current study reported resistance of R. docoloratus larvae when exposed to organophosphate and synthetic pyrethroids with no resistance were reported on the amidines. These findings concur with the report by and who found similar results where all the tick larvae did not show resistance during exposure to the amidines. It was further argued that even though the three acaricide groups have been used over the years, however, amidines has been not commonly used in high tick population in which that lowers chances for tick’s selection pressure compared to organophosphates and synthetic pyrethroids [31]. It is further anticipated to that amidines effectively control both multi-host ticks together with single-host ticks [32]. Of the three localities, all tested R. docoloratus larvae did not show resistance when exposed to all the acaricides used for testing in Senqu region. This was attributed to the fact that there was well-developed ticks control program at Senqu in which it was compulsory to every cattle farmer to bring the cattle in the dipping tank of which suppressed tick’s population in the region and subsequently making difficult for ticks to develop resistance. The majority of the cattle farmers indicated that they increase acaricide concentration during high tick season resulting in complete mortalities after acaricide application and only left with residues [9]. Amongst other factors for tick resistance, the use of the acaricide over a long period of time has been the major contributing factor towards the larvae resistance to organophosphates and synthetic pyrethroids [6,33]. However, even though organophosphates and synthetic pyrethroids showed resistance, findings [5]. From the study that was conducted by [7] suggested that the two acaricides effectively decreases tick population when properly applied to the animal. Thus, therefore, tick’s resistance emergence in R. decoloratus shall encourage proper tick control programs in the regions to mitigate chances of complete resistance to the commonly used chemicals [34].

This study documented R. decoloratus larvae resistance to organophosphate and synthetic pyrethroids except for amidines. The findings from this study concluded that there is a need for a specific focus on the proper application of the acaricides. It was observed that poor acaricide application can lead into selection pressure thus increase incidence of tick resistance [35-37]. It is recommended that future strategy should be developed on the use of acaricide application guided by tick dynamics, as tick population differs from region to region based on the host availability and vegetation. Furthermore, dilution of the acaricides should be guided by the manufactures recommendation and by the trained individual and rotation of the acaricides should be practiced using deferent modes of action.

With this study we would like to acknowledge farmers in Joe Gqabi District Department of Rural Development and Agrarian Reform in Joe Gqabi for their assistance during data collection and farmers to allow us to use their cattle. We further would like to acknowledge National Research Foundation for funding this study.

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