# Bottle-Acaricide-Impregnation-Test (BAIT): an alternative novel bioassay for diagnosis of acaricide tick-tolerance and/or resistance

Charles Junior Ndawula, Fred Sempijja, Patrick Emudong, Nelson Muwereza, Patrick P'Odyek Abila, Rogers Kalyetsi

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## To cite this version:

Charles Junior Ndawula, Fred Sempijja, Patrick Emudong, Nelson Muwereza, Patrick P'Odyek Abila, et al.. Bottle-Acaricide-Impregnation-Test (BAIT): an alternative novel bioassay for diagnosis of acaricide tick-tolerance and/or resistance. Acarologia, 2025, 65 (2), pp.575-583. 10.24349/jglt-6lu6 hal-05122190v2

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# Bottle-Acaricide-Impregnation-Test (BAIT): an alternative novel bioassay for diagnosis of acaricide tick-tolerance and/or resistance

Charles Junior Ndawula ID a,b, Fred Sempijja c,d, Patrick Emudong a,b, Nelson Muwereza ID a,b, Patrick P'Odyek Abila ID a,b, Rogers Kalyetsi ID d

## Original research

### ABSTRACT

**Background:** Ticks are blood-feeding ectoparasites which transmit pathogens ranging from protozoa, viruses, bacteria to both humans and animals. For decades, acaricides have significantly contributed to reduction of the tick-population, and tick-borne diseases. However, there is an increase in acaricide tick-resistance globally. Since the 1960s, diagnosis of acaricide-resistant ticks relies mainly on two methods: the Larval Packet Test (LPT) and the Adult Immersion Test (AIT). The limitations of these methods are for instance, (1) each assay is restricted to one stage of tick-development. (2) the assays require special additional reagents and materials therefore unaffordable in resource constrained regions. **Objective:** To address these gaps, we have developed an alternative novel method named: the Bottle-Acaricide-Impregnation-Test (BAIT) and assessed its efficacy. **Methodology:** We examined the potential of BAIT to detect acaricide resistant and/or tolerant ticks. Three acaricides: DuoDip (Organophosphate), Norotraz (Amidine) and SyperTix (Synthetic pyrethroid) were independently tested against unfed and fed *Rhipicephalus appendiculatus* colony ticks at different stages of development (larvae, nymphs and adults). The test acaricides were applied separately at single (1x) and double (2x) working concentration and the efficacy of the BAIT assessed in comparison with the AIT, LPT and Nymph Immersion Test (NIT). **Results:** Our data reveals that: (1) the BAIT assay is as efficient as the AIT, LPT, and NIT, and (2) the efficiency of the tests does not vary with acaricide class or concentration (3). **Conclusion:** The BAIT method is universal to all tick-developmental stages, simpler, cheaper and could be applicable both under field and laboratory conditions.

**Keywords** acaricides; resistance; ticks

### Introduction

Ticks are blood-feeding ectoparasites capable of transmitting a broad range of pathogens, including bacteria, protozoa, viruses, nematodes, and fungi to humans and animals (de la Fuente et al. 2008). Tick-borne-disease (TBD) sustainable-control is attainable mainly through chemoprophylaxis and vaccination. However, due to the complexity of tick-borne pathogens, TBD-specific vaccines are elusive, yet chemoprophylactic drugs are expensive. Consequently, reduction of the animal-tick burden using acaricides remains the first line for control of TBDs (Rodriguez-Vivas et al. 2018). Although there are several ways of applying acaricides,

Received 14 February 2025
Accepted 27 May 2025
Published 11 June 2025

Corresponding author
Charles Junior Ndawula ID:
ndawulaj821@gmail.com

Academic editor
Marčić, Dejan

https://doi.org/10.24349/jglt-6lu6

ISSN 0044-586X (print)
ISSN 2107-7207 (electronic)

<figure><img src="image_3.png" /></figure>

Ndawula C. J. et al.

Licensed under
Creative Commons CC-BY 4.0

<footnote>a National Agricultural Research Organization, P.O Box 295, Entebbe, Uganda.</footnote>

<footnote>b National Livestock Resources Research Institute, P.O Box 5704, Wakiso, Uganda.</footnote>

<footnote>c Uganda institute of Allied health and management Sciences-Mulago P. O Box 34025, Kampala, Uganda.</footnote>

<footnote>d Mbarara University of Science and Technology P.O Box 1410, Mbarara, Uganda.</footnote>
such as spraying, dipping, and pour-on (Moraes et al. 2023), all methods aim to impregnate
the acaricide onto the animal skin, which serves as a tick-decoy. Similarly, Davidson and
colleagues (2024) have explored this approach in developing acaricide impregnated military
uniforms toward control of human tick bite exposure. However, the prolonged and improper
use of acaricides has led to the emergence of acaricide-resistant tick populations (Rodriguez-
Vivas et al. 2018), thereby downplaying the significance of acaricides in TBD-control.
Therefore, regular surveillance of the ticks' acaricide susceptibility is critical in determining
the acaricide withdraw period and the effective acaricides. Until now, researchers have
illustrated several assays for monitoring the acaricide resistant tick populations. These include
the Larval Immersion Test (LIT) (Shaw 1966), Larval Tarsal Test (LTT) (Lovis et al. 2011),
Syringe immersion test (SIT) (Shindhu et al. 2012), Nymph Immersion test (NIT) (Baker
and Shaw 1965), and RaTexT® (Jongenjan et al. 2024). However, the Food and Agricultural
Organization (FAO 2004) recommends only two methods: Larval Packet test (LPT) (Stone
and Haydock 1962), and the Adult Immersion test (AIT) (Drummond et al. 1973). However,
these methods have several drawbacks. For instance, (1) both LPT and AIT undermine the
fundamental biological differences between one-host and multiple-host ticks. In particular,
whereas the entire life cycle of the one-host ticks, such as *R. microplus*, prevails on the cattle
for 30 days (Senbill et al. 2018), at each stage of the life cycle, multiple-host ticks, such
as *R. appendiculatus*, stay on the animal for fewer days, feed to engorgement and detach to
the ground (Troughton and Levin 2007). The implication is that, in contrast to multiple-host
ticks, one-host ticks are consistently exposed to acaricides. Therefore, the accuracy of the
acaricide bioassay could vary between the tick life cycle stages of the three-host ticks versus
one-host tick. Additionally, differences in response to acaricides have also been observed
between engorged (blood-fed) and unfed ticks (Delmonte et al. 2017). (2) assembly of the
LPT assay requires specific additional reagents and materials, such as olive oil, bull-dog
clips, Whatman filter paper (grade 1), and Trichloroethylene (FAO 2004), making the assay
costly. These drawbacks highlight the need for novel methods which are simpler, cheaper, and
universal to all tick life cycle stages. This research addresses FAO's (2004) long-standing call
for the development of novel diagnostic tools to detect acaricide resistance in ticks that are
affordable in resource-limited areas. Therefore, we propose an alternative novel assay, the
Bottle-Acaricide-Impregnation-Test (BAIT). The proposed assay mimics the CDC bottle assay
(Brogdon and McAlister 1998), but with modifications. Unlike the CDC bottle assay, the BAIT
assay exploits the presence of the surface-active agent (surfactant) in the acaricide formulation.
Similar to plant pesticides (Parr and Norman 1965), the surfactant promotes the reduction of
the acaricide surface tension, leading to increased surface adsorption of the acaricide. Our
hypothesis was that acaricides can independently adsorb onto other surfaces, such as plastic
bottles, therefore, can be used for examining acaricide tolerance and/or resistance of ticks. This
study addressed four fundamental research questions: (1) Is the BAIT assay as efficient as
the currently FAO (2004) recommended methods (LPT and AIT)? (2) Is the BAIT-detection
efficacy influenced by the class of acaricides? (3) Is the efficacy of the BAIT assay dependent
on acaricide concentration? (4) Is the BAIT assay applicable to all stages of the tick's life
cycle (larvae, nymphs, and adults)? Therefore, the goal of this study was to investigate the
efficacy of the BAIT-assay in diagnosing acaricide resistant and/or tolerant tick-populations.
The development of the novel alternative assay involves the impregnation of plastic sample
bottles with selected types of acaricides from different classes diluted at single (1x) and double
(2x) working concentrations. The acaricide-impregnated bottles were used to examine acaricide
resistance among laboratory-bred *Rhipicephalus appendiculatus* ticks. The BAIT method was
examined in comparison with the FAO (2004) recommended methods: LPT, NIT, and AIT.
This novel assay could be applied globally for (1) laboratory and field conditions (2) diagnosis
of acaricide-resistant or tolerant tick populations (3) assessing the efficacy of new acaricide
formulations. Ultimately, diagnosis of acaricide resistant and/or tolerant tick populations
guides farmers and decision makers; on the acaricide withdraw period and on the choice of the
appropriate effective alternative acaricide(s).
# Materials and Methods

## Ethical review

The study protocol was reviewed by the Research Ethical Committee and the Faculty Research Committee of the Faculty of Medicine, Mbarara University of Science and Technology (Reference: MUST 2023-1166).

# Materials and Methods

## Ticks

The *R. appendiculatus* ticks used in this study were collected from Nakasongola district which is located in the cattle corridor of Uganda. Farmers in Nakasongola use different acaricides for tick control namely Amitraz, DuoDip (data unpublished). The ticks used in this study were maintained in the colony (Bailey 1960) at NaLIRRI-NARO for up to five generations without exposure to acaricides.

## Chemicals

Three classes of acaricides: DuoDip 55% E.C. (organophosphate), Norotraz 12.5% E.C. (amidine) and SyperTix 10% E.C. (synthetic pyrethroid) were used for all assays. These represent the classes of acaricides used by the farmers in the study area. The acaricides were obtained from a farm supply shop and were certified by the National Drug Authority of Uganda. All the acaricides were independently diluted to single (1x) and double (2x) working concentration. At 1x concentration, the acaricide was diluted as recommended by the manufacturer whereas at 2x the acaricide recommended concentration was doubled.

## Consumables

Magnifying lens, tally counter, white tray, white paper (A4 size), masking tape, fine art soft brushes, petri dishes (5 x 5 cm), forceps, olive-oil, trichloroethylene, bull dog clips, Whatman filter (grade 1, 25mm) paper, plastic sample bottles (100 ml) and cut pieces of nylon cloth.

# The Bioassays

## The Larval and Nymph packet Test

The LPT and NIT assay was conducted as described by Stone and Haydock (1962) and by Baker and Shaw, (1965) using unfed larvae and nymphs respectively, but with slight modifications. To prepare the LPT, the acaricides were independently diluted with two parts of Trichloroethylene and one part of Olive oil to single (1x) and double (2x) working concentration. Twenty milliliters of the mixture were dispensed into a petri dish and a filter paper of was immersed for 1 min to allow impregnation. The wet filter paper was left to stand for one hour in a running fume hood to allow air drying. The dried filter paper was folded in the middle and sealed at the sides using bulldog clips after which the unfed larvae were added. To prepare the NIT, the acaricides were diluted in water to single (1x) and double (2x) working concentration. Thereafter, the unfed nymphs were immersed into 5 ml of the diluted acaricide for one minute, removed, dried on a paper towel and finally transferred to the packet made-up of non- impregnated filter papers. For each assay, approximately, 100 active unfed larvae or nymphs were counted and gently dispensed into the packets and sealed using a third clip. The tick-packets was placed in an incubator at 27 °C, 85-90% RH and the acaricide-induced mortality was assessed after 24 h based on which the efficacy of was determined as described in the formular below.

## Adult Tick Immersion test (AIT)

The AIT assays were conducted following the FAO, (2004) guidelines. The acaricides were independently diluted with water to 1x and 2x working concentration. The adult female unfed
ticks were immersed into the test-acaricide formulation in a petri dish and let to immerse for 30 minutes with periodical agitation after every 10 minutes. Then the immersed ticks were transferred onto an acaricide-free dry paper towel. The dry ticks were placed into acaricide-free sample bottles, incubated at 27 °C, 85-90% RH and the acaricide effect assessed after 24 h. All assays were performed in triplicate for each test acaricide. Additionally, following the procedure described by Drummond and colleagues (1973) AIT assay was performed to assess effect of the acaricides on 10 engorged adult female ticks.

## The BAIT assay for Larvae, Nymph and Adults

All acaricides were independently diluted in water to 1x and 2x working concentration Five milliliters of the diluted acaricide were dispensed into the new 50 ml polypropylene sample bottles (regularly used in hospitals for urine sample collection), covered with a nylon cloth (0.8- 1 mm mesh size), tilted at 45° while swirling for 5 min, after which the bottle was let to stand for 5 min. This was repeated three times to ensure total drenching and impregnation of the sample-bottle interior surface. Thereafter, the test-acaricide was discarded, the impregnated sample-bottles were thoroughly drained and let to air-dry for 1 hour at room temperature. At least 100 unfed larvae, nymphs or adults were counted and transferred to the acaricide impregnated bottles. All assays were performed in triplicates. Additionally, the efficacy of the BAIT assay against engorged female ticks was assessed following the same procedure but with a slight modification. Particularly in this case, because the engorged ticks are bigger than the unfed ticks, a bigger surface area is required to ensure tick-total contact with the surface of the product impregnated container. Therefore, petri dishes were used to ensure uniform acaricide-tick exposure and separation of the ticks to track their individual ability to lay eggs. After exposure to the acaricide impregnated petri dish, the ticks were assessed as described by (Drummond et al. 1973; FAO 2004).

## Data collection and analysis

### Assay efficacy assessment

#### Tick mortality

The assay-efficacy against the unfed ticks (larvae, nymph and adults) was assessed 24 h after test-acaricide exposure based on the acaricide-induced tick mortality. The acaricide-induced mortality was depicted based on the number of the immobilized ticks.

#### Tick resistance

The assay-efficacy against engorged adult female ticks was assessed 7 days post exposure. The percentage resistance was determined based on tick-egg laying ability of the acaricide-treated ticks versus the untreated ticks (FAO, 2004). The percentage resistance is calculated as indicated below:

$$ \text{Resistance (\%)} = (Nt/Nw) \times 100 $$

Nt is number of treated ticks laying eggs

Nw is number of untreated ticks laying eggs

### Statistical Data analyses

Data were subjected to ANOVA at 99.05% (p=0.05) confidence level using R program (R-4.32 version). Where the mean square value was significant, data were further subjected to Post hoc test using Tukey's procedure Shapiro-Wilkson test (P=0.05).

## Results

Based on the unfed-tick mortality rate, the efficacy of the BAIT assay was determined in comparison to the three commonly used assays (LPT, NIT and AIT) using three test acaricides diluted to single (1X) and at double (2x) working concentration. The BAIT assay was as efficient
as the LPT, NIT and AIT (figure 1 A–C). With all the test acaricide, no difference in mortality was noted at single (1x) and double working concentration (p=0.381). A difference in mortality was noted among the three classes of acaricides and at the stage of tick development (larvae, nymph and adult). At the larval-stage, higher mortality was shown with the organophosphate (DuoDip) and Synthetic pyrethroid (SyperTix) acaricide than with the amidine (Nortotraz). A similar pattern was noted at the nymphal and the adult-stage. At all the tick-development stages, Amidines induced the lowest tick-mortality rate. Another research question was; is the BAIT assay also efficient in detecting resistance among engorged/fed ticks? To answer the question, the BAIT assay was compared with the AIT. Similarly, 3 test acaricides were used but at single (1x) concentration. The efficacy was determined based on the egg-laying ability post acaricide exposure.

Our data (Table 1) revealed difference between the efficacy of the BAIT and the AIT assay with different test acaricides. Both assays were equally efficacious with synthetic pyrethroids (SP) whereas with amidines (AM) and organophospates (OP), AIT was more efficacious than BAIT assay.

Discussion

Ticks are ranked second to mosquitoes in pathogen-transmission, because they are capable of transmitting pathogens ranging from viruses, bacteria, protozoa, to humans and animals (de la Fuente *et al.* 2008). One of the key factors that foster tick-pathogen transmission is the high egg-laying capacity and the ability to thrive under harsh field conditions. For instance, *Ixodid* ticks such as *Amblyomma variegatum*, *R. appendiculatus* lay at least 2000 eggs with over 90% egg viability (Leal *et al.* 2020). Therefore, tick-population reduction using acaricides remains fundamental in tick-borne disease control. However, after decades of acaricide application, populations of acaricide-resistant ticks have emerged globally (Rodriguez-Vivas *et al.* 2018).

Since the 1960s, detection of the acaricide-resistant tick population has relied on two FAO recommended methods: LPT and AIT (FAO 2004). Although the two methods are widely used, they have limitations. For instance, each method is specific to a particular tick stage of development, and both are limited to laboratory conditions. This study illustrates a novel alternative method for detecting acaricide resistant/ tolerant tick population; the Bottle Acaricide Impregnation Test (BAIT). The BAIT assay is similar to the CDC bottle assay that was developed by Brogdon and McAlister (1998). However, several modifications were made in the BAIT assay. For instance, whereas the CDC assay is performed using glass bottles, we opt to use plastic bottles which are cheaper and readily available. Additionally, unlike with CDC assay, the BAIT assay exploits the properties of acaricide formulation without addition of surface binding synergists. Therefore, while preparing the BAIT assay only was water added to the acaricide as per the manufacturer's instructions.

<table><caption>**Table 1** Examining the BAIT assay efficacy in comparison with the AIT assay against engorged adult ticks using three test acaricides classes Amidine (AM), Organophosphate (OP), and Synthetic pyrethroid (SP) each at single (1X) concentration and water.</caption><thead><tr><th>Method</th><th>Treatment</th><th>Conc</th><th>No. Treated-ticks</th><th>No. laying-Ticks</th><th>% Resistance</th></tr></thead><tbody><tr><td>AIT</td><td>Water</td><td>0</td><td>10</td><td>10</td><td>100</td></tr><tr><td>AIT</td><td>OP</td><td>1x</td><td>10</td><td>0</td><td>0</td></tr><tr><td>AIT</td><td>AM</td><td>1x</td><td>10</td><td>0</td><td>0</td></tr><tr><td>AIT</td><td>SP</td><td>1x</td><td>10</td><td>10</td><td>100</td></tr><tr><td>A-BAIT</td><td>Water</td><td>0</td><td>10</td><td>10</td><td>100</td></tr><tr><td>A-BAIT</td><td>OP</td><td>1x</td><td>10</td><td>2</td><td>20</td></tr><tr><td>A-BAIT</td><td>AM</td><td>1x</td><td>10</td><td>10</td><td>100</td></tr><tr><td>A-BAIT</td><td>SP</td><td>1x</td><td>10</td><td>10</td><td>100</td></tr></tbody></table>
<figure><img src="image_4.png" /><figcaption>**Figure 1** Examining the Bottle Acaricide Impregnation Test (BAIT) assay efficacy against Larvae (A), Nymph (B) and Adults (C) respectively with reference to the conventional assays: Larval packet Test (LPT), Nymph Immersion test (NIT) and Adult Immersion Test (AIT). The efficacy was determined based on the mortality rate of unfed larvae, nymphs and adult *Rhipicephalus appendiculatus* ticks after a 24 h exposure to different acaricides diluted to single (1X) and at double (2x) working concentration. The classes of the acaricides are depicted as Amidine (AM), Organophosphate (OP), and Synthetic pyrethroid (SP) and are tested in reference to the control (CONT). The BAIT assay is shown to be as efficient as the LPT, NIT and AIT assay.</figcaption></figure>

We also examined whether doubling the acaricide working-concentration could impact the BAIT method proficiency. Note that this mimics one of the common practices applied by farmers to enhance the efficacy of acaricides (Mugabi *et al.* 2010). On comparing the BAIT assay, with the LPT, NIT and AIT, we established that there was no significant difference in efficiency (figure 1 A–C) at both acaricide working concentrations (1x and 2x). The proficiency of the BAIT method could be attributed to presence of surfactant within the acaricide formulations which promotes lowering of the acaricide surface tension and formation of the surface film leading to: (1) increase in acaricide surface-dispersion (2) reduction of
the acaricide wash-down. The role of surfactants is extensively discussed, for instance, in formulation of plant pesticides (Parr and Norman, 1965). Considering that acaricides constitute surfactant(s), addition of trichloroethylene and olive oil while preparing the LPT assays does not impact on the proficiency of the test, hence the additional chemicals are redundant. Instead, adding other chemicals increases the cost of the methods, and yet for instance, trichloroethylene is carcinogenic to the end user (Chiu *et al.* 2013). Despite their proficiency, the currently used methods (LPT, NIT and AIT) are restricted to a specific stage of tick development whereas the BAIT method is universal to all the stages. Additionally, the LPT, AIT and NIT require use ticks with a different feeding status. However, while developing the LPT versus AIT and NIT, the researchers neglected the physiological differences among the fed or unfed ticks and at different stages of development. For instance, differences in gene transcript expression have been revealed between fed and unfed ticks (Charrier *et al.* 2018; Ring *et al.* 2022). The acaricides could also influence the gene transcripts among fed and unfed ticks. We noted an inconsistency in response among the fed (engorged) and unfed adult ticks using the AIT and A-BAIT method (Table 1) (figure 1C). Similarly, Delmonte and colleagues (2017) illustrated differences in response to acaricides among fed and unfed nymphs. These findings emphasize the potential role of physiological differences in acaricide response; an aspect neglected by the currently recommended methods (FAO, 2004): LPT (Stone & Haydock, 1962) and AIT (Drummond *et al.* 1973). Our findings suggest concern over the accuracy of the current AIT method. We suggest that the BAIT method is conducted using unfed ticks (larvae, nymphs, adults). However, if the users intend to use engorged ticks, calculations should take the acaricide response against the emerging unfed larvae into account. The reason is that, as often shown in other drug-resistant biological organisms, the gene(s) coding for acaricide tick resistance is hereditable (Rodriguez-Vivas *et al.* 2018).

In addition to illustrating an alternative novel method for examining acaricide resis-
tant/tolerant population, this study highlights the impact of concentration on acaricide potency.
Doubling the acaricide concentration is a common practice used by farmers in desperate search
for ways to kill acaricide resistant/ tolerant ticks (Mugabi et al. 2010). However, our findings
show that doubling the acaricide concentration does not lead to increased acaricide potency,
hence not relevant in controlling the resistant tick population.

Finally, whereas the study aim was to compare the efficacy of the BAIT assay versus the
current assays (AIT, LPT, NIT), our data reveal difference in acaricide potency. This trend
could be attributed to a difference in the active chemical ingredient. For instance, unlike
the other test-acaricides (Norotraz and Sypertix), DuoDip consists of two active chemical
ingredients: Chlorpyrifos and Cypermethrin (as per the manufacturers' description) explaining
why it is more lethal than other acaricides.

**Conclusion**

This study demonstrates an alternative novel bioassay for detection of acaricide tick resistance and/or tolerance. In addition to showing an no significant difference in proficiency with the FAO recommended methods (LPT and AIT), the BAIT method has several merits over the other assays. (1) The novel method is less costly especially because apart from the plastic sample bottles no other chemicals and consumables are required. Moreover, because of the acaricide-surface adsorption effect, once impregnated, the novel assay bottles could be re-used without re-addition of the test acaricide (data unpublished). (2) It is applicable to all stages of the tick life cycle and potentially to all tick species. (3) It is applicable both under field and laboratory conditions. (4) It does not require highly trained personnel. Ultimately, because of the aforementioned properties, the BAIT method simplifies diagnosis of the acaricide tick resistance/ tolerance, and it is affordable to the resource-limited areas, hence suited for global use.
Role of the funding source

This article has been produced with the financial assistance of the European Union (Grant no. DCI-PANAF/2020/420-028), through the African Research Initiative for Scientific Excellence (ARISE), pilot program. ARISE is implemented by the African Academy of Sciences with support from the European Commission and the African Union Commission. The contents of this document are the sole responsibility of the author(s) and can under no circumstances be regarded as reflecting the position of the European Union, the African Academy of Sciences, and the African Union Commission.

Acknowledgments

We thank (1) Dr. Dania Richter of the Technische Universität Braunschweig, Institute of
Geoecology, Landscape Ecology & Environmental Systems Analysis for the support offered
during preparation of the manuscript. (2) Mr. Omwene E, Mr. Mayeku M and Mr. Omuno E
for the support offered in maintaining the tick colony and assessing the efficacy of the novel
assay. (3) The National Livestock Resources Research Institute which is under the National
Agricultural Research Organization for hosting this research study.

ORCID

Charles Junior Ndawula https://orcid.org/0000-0002-4978-936X

Nelson Muwereza https://orcid.org/0009-0004-4109-7299

Patrick P'Odyek Abila https://orcid.org/0000-0002-4487-1217

Rogers Kalyetsi https://orcid.org/0000-0002-4232-7875

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