Bioassay device and method

Abstract

A bioassay device for determining acaricide resistance by ticks comprises at least one multi-cell structure (1...3, 5...7) of a first cell (11) for containing one or more sample ticks and a group of further cells (12...14) for containing one or more sample ticks. The cells (11...14) are mutually isolated from one another. Said first cell (11) is void of said acaricide to be tested, while said further cells (12...14) comprise said acaricide to be tested in increasing concentrations. Equal numbers of, particularly eight, semi-engorged ticks are distributed over the cells of said at least one multi-cell structure and a mortality per cell is determined after dark exposure of said ticks at room temperature under moist conditions.

Description

[0001] Bioassay device and method

[0002] The present invention relates to a bioassay device for determining acaricide resistance by ticks, particularly ixodidae, comprising at least one multi-cell structure for containing one or more sample ticks, comprising a first cell and a group of further cells, said cells being mutually isolated from one another, wherein said first cell is void of said acaricide, and wherein said further cells comprise said acaricide to be tested in increasing concentrations. The invention further relates to a method of using such a bioassay device.

[0003] Ticks and tick-borne diseases rank high in their impact on animal and human health worldwide. In livestock, tick-borne diseases caused by pathogenic protozoa and rickettsiae constitute a significant constraint to animal production, particularly in tropical and subtropical areas. A relatively small number of tick species infesting livestock and companion animals have developed resistance to acaricides due to their life cycle and ability to build up large numbers on their preferred hosts. This concerns, for example, Rhipicephalus tick species, particularly Rhipicephalus decoloratus, Rhipicephalus microplus, Rhipicephalus appendiculatus, and Rhipicephalus sanguineus on cattle and dogs.

[0004] Rhipicephalus decoloratus, also called the African blue ticks, are small and inconspicuous, with short mouthparts and slender legs. The brownish-yellow males are considerably smaller than the females and their darker-coloured intestine is visible through the lightly sclerotised scutum. They can generally be found paired with the females. The engorged females are bluish-brown and attached mainly to the cattle's face, neck, shoulders and escutcheon. Rhipicephalus decoloratus prefers cattle as hosts. In addition to cattle, it frequently parasitises horses, donkeys, sheep, and goats.

[0005] Rhipicephalus decoloratus has a one-host life cycle, where moulting from larva to nymph and nymph to adult occurs on the host. The time spent on the host, from the attachment of the unfed larva until detachment of the engorged female, is approximately three weeks; because of its one-host life cycle, all stages of development occur simultaneously on the host, building up to a large tick burden. This tick is distributed throughout most of sub-Saharan Africa, except for areas where B. microplus have replaced it.

[0006] Rhipicephalus microplus, also called the Asian blue tick, is slightly larger than R. decoloratus and somewhat more red but is otherwise very similar in appearance. In sub-Saharan Africa, R. microplus has a one-host life cycle on cattle and, like R. decoloratus, can complete more than one generation in a year. Probably R. microplus was introduced into East and South Africa from Madagascar, where it initially arrived with southern Asian cattle. Where favourable moist and warm climatic conditions exist, it competes with and can replace the indigenous R. decoloratus.

[0007] The known distribution and ecological preferences of the blue cattle ticks, have dramatically changed after R. microplus was introduced inadvertently with imported Brazilian cattle into West Africa (Ivory Coast) and in the subsequent years quickly spread further throughout the region. The spread of R. microplus ticks on cattle has replaced most indigenous R. decoloratus populations in large geographical areas throughout West, East and Southern Africa. Because this tick transmits both Babesia bovis and Babesia bigemina, it poses a more significant potential threat to livestock production than R. decoloratus.

[0008] Rhipicephalus appendiculatus is a medium-sized brown tick with short mouthparts. The legs of the males increase markedly in size from the first to the fourth pair, and engorged males have a slender caudal process. Adults parasitise medium-sized to large ruminants, while the immature stages feed on most ruminant species and other mammals, including equids, carnivores and hares. Among domestic animals, cattle are the preferred hosts and can become very heavily parasitised at all stages of development. Adults attach in the highest numbers to the ears of their hosts but are also found on other parts of the body. Rhipicephalus appendiculatus is an eastern, central, and southern African tick and the primary vector of Theileria parva that causes East Coast fever. Rhipicephalus sanguineus, also called the brown dog tick, is vector of a range of bacterial and protozoan pathogens adversely affecting canine health in many (sub) tropical parts of the world. With all stages feeding on dogs, the three-host life cycle of Rhipicephalus sanguineus, like Rhipicephalus linnaei, can lead to an uncontrolled build-up of large tick populations.

[0009] Tick control on livestock heavily depends upon applying commercial acaricidal products to livestock and companion animals. However, ticks can resist acaricides when repeatedly exposed to the same chemicals applied to livestock or companion animals. Acaricide resistance in ticks is a significant concern for the livestock industry in Latin America, Asia, Australia, and sub-Saharan Africa. Acaricide resistance in cattle ticks is a significant concern for livestock in (sub)tropical regions, where resistance is also emerging in ticks infesting dogs.

[0010] Ticks are among the most important vectors of diseases affecting livestock, companion animals, and humans. Tick-borne diseases rank high in their impact on animal and human health worldwide. In livestock, tick-borne diseases caused by pathogenic protozoa and rickettsiae constitute a significant constraint to animal production, particularly in tropical and subtropical areas. Companion animals, particularly dogs, pay a heavy toll on tick-borne diseases, such as babesiosis and ehrlichiosis.

[0011] Rhipicephalus microplus and Rhipicephalus decoloratus are among cattle's most damaging tick species. Blue ticks have a one-host life cycle, which allows them to develop resistance faster since they are exposed to acaricide-treated cattle for almost three weeks. Resistance in cattle ticks has been reported against all acaricidal classes, leading to global economic losses. The basic mechanisms that underlie resistance are target-site mutations, increased metabolic detoxification, and reduced cuticular penetration of the tick's cuticula. Resistance of ticks to acaricides is recognised as a failure of treatment to eliminate tick burdens from livestock. Although treatment failure often results from incorrect application of the acaricide or substandard acaricide products, the persistence of ticks after correctly applied treatments indicates that resistance has developed.

[0012] The Food and Agriculture Organization (FAO) recommends using the Larval Packet Test (LPT) as a standard tick resistance laboratory test for resistance diagnosis. The Larval Packet Test is, therefore, the most widely used bioassay for detecting acaricide resistance. The Larval Packet Test employs serial dilutions of acaricides impregnated into Whatman filter papers in triplicate. Tick larvae are exposed to these chemically impregnated filter papers in this test, and their subsequent mortality is quantified.

[0013] Although the Larval Packet Test provides a reliable bioassay for determining acaricide resistance by ticks, it is limited by the time it takes. Engorged female ticks must be collected, and female ticks' weight must be above a certain threshold for producing eggs. Proper guidance is required in collecting, handling and submitting engorged ticks to diagnostic laboratories. For most tick species, incubation conditions for engorged female ticks for eggs and larval production should be 27°and 85 % relative humidity. In total, the test takes at least six weeks before testing is completed with larvae derived from engorged female ticks that were collected from animals in the field. Hence, the Larval Packet Test requires laboratory tick-rearing facilities and turns out to be a relatively labourious bioassay because it uses a full range (n=7) of concentrations to cover a 0-100% mortality of larvae.

[0014] As a result, when results reach the owner of the animals, they are of no value in recommending the solution of choice as the next generation of ticks has replaced those on which the resistance test was conducted. Hence, disputes between consumers and producers about product efficacy are complex to settle since there is no means to differentiate resistance from malpractices in chemical tick control.

[0015] The present invention has inter alia for its object to provide a bioassay and related method to determine tick sensitivity or resistance to acaricidal products that allow for pen-side testing of adult ticks immediately after removal from their hosts. To achieve said object, a bioassay device for determining acaricide resistance by ticks, particularly ixodidae, as described in the opening paragraph, according to the invention is characterized in that said first cell is empty or comprises a first impregnated piece of absorbing tissue , in that said further cells each comprise a further impregnated piece of absorbing tissue, in that said first impregnated piece of absorbing tissue is impregnated with a diluent only, and in that said further impregnated pieces of absorbing tissue comprise said diluent with said increasing concentrations of an acaricide to be tested.

[0016] The tissue inside the cells mimic the natural skin of the host animal of the ticks. As a result they will show their normal natural behaviour. Especially when used in conjunction with semi-engorged ticks, this will stimulate the ticks to walk over the tissue inside the cells that release the impregnant. Concurrently the ticks take up the diluent with or without an amount of the acaracide to be tested, depending on the cell. This mimics the way ticks are exposed to acaracides on animals. Especially the still greedy semi-engorged ticks will take up a considerable and possibly already lethal amount of the impregnant in relatively short time. This way of self-exposing the ticks to acaracides is an entirely new approach in the search for a quick and reliable test method.

[0017] A method of using said bioassay according to the invention is characterized in that equal numbers of semi-engorged ticks are distributed over the cells of said at least one multi-cell structure, particularly an equal number of five to eight ticks in each cell, and a mortality per cell is determined after dark exposure of said ticks at room temperature under moist conditions. Using more ticks per cell will deliver a more precise statistical analyses and outcome; fewer ticks may be used to suit local tick availability.

[0018] According to the invention, semi-engorged adult ticks are exposed to acaricidal compounds immediately after being removed from cattle or dogs in the field. The removed ticks are confined to the cells of said multi-cell structure. A few serve as a reference population in the first cell and the remainder is exposed in the further cells to various concentrations of an acaricide to be tested. The test is based on the principle that recently removed partly engorged ticks remain actively walking around to seek to finish their blood meal. During this process, they are exposed to lethal concentrations of an acaricidal compound in the further cells. This type of exposure mimics the behaviour of ticks that embark on an acaricide-treated host. Low, moderate or high acaricide resistance levels might be measured within only hours of tick exposure on site. Susceptible ticks, for instance, may die as soon as three hours after exposure to synthetic pyrethroids. The bioassay according to the invention differentiates resistant adult ticks from susceptible ticks and can potentially replace current labourious tests based on the use of larval ticks.

[0019] When partly engorged adult ticks are collected from their animal hosts and immediately exposed to the acaricide-treated cells, they remain actively walking around to seek to finish their blood meal. This type of exposure mimics the behaviour of ticks that embark on an acaricide-treated host. Ticks are preferably exposed at room temperature in the dark under humid conditions. Each cell is examined by the naked eye to count the dead / alive ticks. The criterion for mortality is the inability of ticks to walk, particularly after some carbon dioxide stimulation for instance by blowing into the compartment. If susceptible, ticks will succumb after exposure to the recommended concentration. If resistant, ticks will survive various acaricidal concentrations. The percentage of knocked-down ticks reflects low, moderate, or high-level resistance to the subject acaricide compound.

[0020] In a preferred embodiment the bioassay device according to the invention is characterized in that said at least one multi-cell structure comprises at least one further multi-cell structure comprising a further group of further cells containing further impregnated pieces of absorbing tissue comprising said diluent with increasing concentrations of a further, different acaricide to be tested. The various multi-cell structures allow for testing multiple specific acaricides in only a single instance. This will provide insight as to what acaricide will perform best for tick control. In a preferred embodiment, the bioassay device according to the invention is thereby characterized in that said diluent comprises an oily liquid, particularly olive oil. A diluent on an oil basis, particularly pure natural olive oil, does not readily vaporize or otherwise drie out. Hence, the ticks will experience a moist environment all the time that allows them to take of the liquid. Moreover, generally acaracides will easily dissolve in an oily liquid.

[0021] A further embodiment of the bioassay device according to the invention is characterized in that further cells comprise a recommended concentration of the acaricide to be tested and respective multitudes of said recommended concentration. More particularly a further embodiment of the bioassay device according to the invention is characterized in that said multitudes of said recommended concentration comprise five times said recommended concentration and ten times said recommended concentration. In this manner the bioassay according to the invention may be adopted to the resistance intensity protocol from the latest WHO guidelines for resistance detection in malaria mosquito vectors, which uses lx, 5x and lOx higher concentrations, revealing a low, moderate, and high resistance intensity, respectively.

[0022] In view of providing a quick and clear test result at a glance, a further preferred embodiment of the bioassay device according to the invention is characterized in that the cells of said multi-cell structure are arranged in at least one row holding multiple positions, said first cell occupying a first position in said row, and in that said group of further cells is distributed over further positions in said row in an order of increasing acaricide concentration. A column within said row or rows showing adequate mortality, for instance exceeding 90%, immediately indicates a suitable concentration to effectively control tick propagation and proliferation.

[0023] Although a single row suffices to test acaricide resistance of the harvested semiengorged ticks, form a statistical perspective a preferred embodiment of the bioassay according to the invention is characterized in that said row is a first row of a number of substantially identical rows for testing the acaricide resistance of said acaricide, and more particularly in that said multiple-cell structure comprises three substantially identical rows of cells for each acaricide resistance to be determined. In such a case the results of several rows may be averaged to provide a more accurate outcome.

[0024] In a particularly practical and favourable setup, the bioassay device according to the invention is characterized in that at least said further cells of said at least one multi-cell structure are provided by pockets containing said impregnated pieces of said absorbing tissue, said impregnated pieces of absorbing tissue comprising pieces of fabric or non-woven material being arranged inside said pockets. The pockets provide compartments in which the ticks that were caught may be stored and exposed to the acaricide to be tested with various, selected concentrations. The container provides both a storage as well as testing facility for carrying out the bioassay according to the invention.

[0025] The pockets of said container may be arranged in rows, to provide a quick and comprehensive test result, while said container may comprise a distinct row of empty pockets. These empty pockets that do not contain an impregnated piece of absorbing tissue may be used to collect and store completely engorged ticks, left untreated for later research. The pockets of the multi-cell structure, on the other hand, will accommodate semi-engorged tick that are still hungry and prone to digesting the active compound in the impregnated piece of absorbing tissue.

[0026] A distinct row of empty pockets may favourably be used to separate a first multi-cell structure from a further multi-cell structure for testing different acaricides at the same time. To that end, a further embodiment of the bioassay device according to the invention is characterized in that said cells are provided by corresponding rows of pockets of said container, in that said container comprises a first group of mutually adjacent rows of pockets for testing said first acaricide, in that said container comprises a further group of mutually adjacent rows of pockets for testing a further, different acaricide, and in that said first groups of rows is separated from said further group of rows by said distinct row of empty pockets. Although the pockets may be loaded in various ways with an impregnated piece of absorbing tissue that comprises said diluent only or said diluent with concentration of containing the acaricide to be tested, a particularly practical embodiment of the bioassay device according to the invention is characterized in that at least said further cells of said at least one multi-cell structure are provided pockets containing impregnated pieces of an absorbing tissue that are arranged inside said pockets. The pieces of tissue may for instance comprise a woven or non-woven textile, cloth or any other absorbing fabric or material. Particularly a synthetic fabric, for instance a polyamide such as nylon, may be used for said tissue.

[0027] Conveniently the pieces are attached to the bottom of the respective pocket to immobilize it. In order to ensure a proper exposure of the ticks to the impregnated material, a further preferred embodiment of the bioassay is characterized in that said pockets are confined by portion of a bottom of said container and by closed walls that separate said pockets , and in that said impregnated pieces of tissue occupy an majority of said portion of said bottom within a pocket and extend over at least part of a height of said walls.

[0028] In order to avoid the ticks escaping the compartments of the container, a preferred embodiment of the bioassay device according to the invention is characterized in that each pocket is sealable by an individual transparent lid and in that said container comprises a transparent further lid jointly securing the individual lids of the pockets of the container in a closed position. The individual lids may be used to capture the ticks in each compartment, while the joint lid of the container will counteract any of the lids inadvertently opening during transport or handling.

[0029] A practical and low-cost embodiment to that end is characterized in that said container, including said lid, is formed from transparent polypropylene. Due to the use of a transparent material for the lids there is no need of opening of the container for investigation of the test result. For a quick and easy understanding of an outcome of the test, preferably the pockets are labelled according to their acaricide concentration. The bioassay device of the invention may be used to determine the acaricide resistance of adult ticks against all commercially available acaricide. In that respect, the bioassay device according to the invention is characterized in that said acaricide comprises at least one acaricide from a group containing pyrethroids, formamidines, organophosphates, macrocyclic lactones, phenylpyrazoles, and isoxazolines.

[0030] Pyrethroids are sodium channel modulators which cause nerve excitation. They were introduced between 1975 and 1983 and include deltamethrin, (alpha) cypermethrin, flumethrin, permethrin and cyhalothrin. Synthetic pyrethroids have exceptional acaricidal properties and long residual effectiveness. Cypermethrin, deltamethrin, and cyhalothrin are most used to control ticks. Resistance to synthetic pyrethroids was first documented in the late 1980s in Australia and Brazil.

[0031] Formamidine-based acaricides, such as amitraz and cymiazole, exert toxicity on the octopamine receptor. Formamidines were introduced for tick control in the mid-1970s. Amitraz is the most effective for controlling ticks. Its toxicity to cattle and humans is minimal and comparatively less persistent in the environment. However, the first amitraz resistance in ticks was already reported in the early 1980s in Australia.

[0032] Organophosphates are acetylcholinesterase inhibitors and include chlorpyrifos, chlorfenvinphos, diazinon and coumaphos. Organophosphates were introduced in the mid-1950s. They are chemically unstable and non-persistent in the environment. The first documented case of organophosphate resistance was in cattle ticks in the mid-1960s in Australia. Due to their relatively high toxicity to livestock, many products have been withdrawn from the market. However, they are still used in many combination products with active ingredients from other acaricidal classes.

[0033] Macrocyclic lactones were introduced in the mid-1970s to control a wide range of arthropods and nematodes. Particularly doramectin, abamectin, eprinomectin, and moxidectin have acaricidal properties. They are increasingly used in combination products with other classes of active ingredients. Macrocyclic lactones have acaricidal efficacy at low doses and can be administered subcutaneously or in pour-on formulations. The first report of resistance to ivermectin and doramectin in cattle ticks was in 2001 in Brazil and subsequently in Argentina, India, South Africa and Uruguay. More recently, resistance to ivermectin was reported in the brown dog tick Rhipicephalus sanguineus.

[0034] Fipronil is a phenylpyrazole compound used to control tick infestations, primarily on dogs and to a lesser extent also on cattle, was introduced in the mid-1990s. In 2007, the first fipronil-resistant cattle ticks were reported. The extensive use of fipronil on dogs has resulted in resistance of the brown dog tick, Rhipicephalus sanguineus.

[0035] Isoxazolines are specific blockers of ligand-gated chloride channels. Afoxolaner, fluralaner, sarolaner and lotilaner are currently marketed as oral canine products against a range of companion animal tick species. Fluralaner is additionally available as a topical spot-on. Recently, the first fluralaner-based pour-on product against ectoparasites infesting cattle has entered the livestock market in Brazil.

[0036] Although various diluents may be used to prepare a test formulation to be introduced in the impregnated piece of absorbing tissue or tissue, particularly said diluent may comprise a mixture of trichloroethylene or acetone and vegetable oil, particularly olive oil. More particularly, said mixture may comprise trichloroethylene or acetone and said vegetable oil in a ratio of at least substantially 2:1.

[0037] In a first specific embodiment said method of using the bioassay device according to the invention is characterized in that said bioassay device is loaded with at least one acaricide comprising an active compound from a group containing containing pyrethroids, organophosphates, macrocyclic lactones, phenylpyrazoles and isoxazolines, and kept in a closed condition in a sealed enclosure, particularly a zipper bag, containing moisturizing means, particularly a moist paper towel, for 24 hours. This embodiment may be used to determine within 24 hours a resistance by the ticks found on livestock or dogs against acaricides containing as an active compound any of pyrethroids, organophosphates, macrocyclic lactones, phenylpyrazoles and isoxazolines.

[0038] In a second specific embodiment said method of using the bioassay device according to the invention is characterized in that said bioassay device is loaded with at least one acaricide comprising an active compound from a group containing formamidines, and kept in a closed condition in a sealed enclosure, particularly a zipper bag, containing moisturizing means, particularly a moist paper towel, for 96 hours. This embodiment is particularly useful to determine resistance against a formamidine-containing acaricide among the ticks.

[0039] Hereinafter, the invention will be described in further detail with reference to a specific embodiment and an accompanying drawing. In the drawing:

[0040] Figure 1 a specific example of a bioassay device according to the invention;

[0041] Figure 2 a row of four interconnected compartments out of the bioassay device of figure 1; and

[0042] Figure 3 a piece of nylon fabricated to be impregnated and to be applied in each of the compartments of figure 2;

[0043] It is noted that the figures are drawn purely schematically and not necessarily to a same scale. In particular, certain dimensions may have been exaggerated to a more or lesser extent to aid the clarity of any features. Similar parts are generally indicated by a same reference numeral throughout the figures.

[0044] An embodiment of the bioassay according to the invention is shown in figure 1 and consists of a container 10 of transparent polypropylene with seven rows 1..7 of individual pockets that are formed by interconnected compartments 11..14 that are all connected together to create the container 10. The container has typically outer dimensions of 120 x 180 x 30 millimetres and may be sealed by a transparent lid 15. Figure 2 shows one of the rows of the container, comprising four interconnected compartments 11,12,13,14 that are each substantially cubical with sides of 20 millimetres. Each compartment 11..14 may be closed by an individual lid 16 that are secured in a closed position once the lid 15 of the container is closed and locked. Both the compartments as wells as the surrounding portion of the container 10, including the lid 15 are entirely formed from transparent polypropylene or any other suitable, preferably transparent or highly translucent plastic.

[0045] Each compartment 11..14 contains a specially designed absorbent matrix 20, shown in figure 3, that may be impregnated with merely a diluent or with a commercial acaricidal product. The matrix is specifically formed to fit the bottom and the walls of a compartment 11..14. To prepare the matrix, a nylon fabric 25 is cut into a suitable format shown in figure 3 using a laser-driven cutter. The nylon fabric 25 has the following specification: PBN-II 30, 102 gsm (3.0 oy). The required pieces 20 of the fabric 25 are collected in a separate zip bag for each dilution. The required amount of acaricide to reach a lx recommended concentration, plus a 5x and a lOx concentrated dose is calculated for each acaricide.

[0046] Several dilutions are prepared using the calculated acaricide concentration and a mixture (2:1) of trichloroethylene (TCE) or acetone with olive oil as a diluent. Each concentration for six rows per container are prepared as well as the control, using diluent only. The required amount of the solution is added to a zipper bag in which the respective matrix pieces were collected. One piece is thoroughly impregnated with 0.37 ml solution. Start with the control and end with the lOx concentrated dose to prevent contamination. The diluted acaricides in the zip bag containing the pieces of the matrix are properly mixed to ensure complete impregnation.

[0047] The matrix pieces are removed from the zip bag and placed on an aluminium drying tray. Start with the control and end with the highest concentration (lOx dose). The pieces may be dried in a fume hood for at least 30 minutes. One piece of the matrix is placed in each compartment of the container and fix to the bottom with a tiny drop of silicon glue. An appropriately impregnated nylon matrix 20 is fitted into each compartment 11..14 of the container 10 to cover the bottom and four sides of each compartment 11..14, excluding the lid. The inside of the lid remains uncovered. The central row 4 remains empty and untreated and can be used to collect fully engorged ticks. Each row 1..3, 5..7 may now contain one compartment 11 without matrix or a diluent only impregnated matrix (=control), one 12 with the recommended concentration (lx), one 13 with a 5x concentrated dose (5x) and the fourth compartment 14 containing a matrix impregnated with a lOx concentrated acaricidal dose.

[0048] The container holds seven rows 1..7 of interconnected compartments that define two distinct multi-cell structures 1..3,5..7 of three rows each. Both multi-cell structures 1..3,5..7 may be impregnated with a different commercial acaricidal product. Thus, there may be two different acaricides per container or both multi-cell structures may be used for extended testing a single acaricide. In this example, however, the test is done on Deltamethrin in one of the multi-structures 1...3 to determine a resistance to this acaricide by adult Rhipicephalus ticks harvested from livestock. Accordingly, in this example, each row contains one compartment 11 without Deltamethrin (=control), one 12 with a recommended concentration 0,25 mg / ml of Deltamethrin (lx), one 13 with a 5x concentrated dose (5x) of 1,25 mg / ml and the fourth compartment 14 containing a matrix impregnated with a lOx concentrated acaricidal dose of 2,5 mg / ml. The compartments within each row 1..3 are labelled C, IX, 5X, 10X respectively as shown in figure 2. The container 10 further comprises a central row 4 of compartments that remain untreated and can be used to collect fully engorged ticks.

[0049] Five to eight partly engorged female ticks, which can walk, are placed into each compartment 11..14 with a pair of tweezers. Start with the control 11 and end with the compartment 14 having the highest acaricide concentration (lOx). The compartments are closed by their individual lids 15 and finally secured in their closed positions by the overall lid 16 of the container 10 that is locked in position by suitable snap members 17 that snap into corresponding recesses or trenches of the lid 16 . The closed container 10 is then placed in a clean opaque zip bag together with a piece of moist tissue paper. The bag is finally zip-sealed completely and left at room temperature for 24 hours. After 24 hours, the container is removed from the bag and the compartments are opened to count live and dead ticks. Start with the control and work up to the highest acaricide concentration. A tick may be considered dead or 'knocked down' if it cannot walk. The collected data are entered into a dedicated Data Capture Sheet, like for instance as shown in table 1 below. Finally all ticks are discarded in a zip bag containing ethanol.

[0050]

[0051] Table 1 RC = Recommended Concentration

[0052] The percentages of knocked-down ticks are calculated to determine whether the ticks are susceptible, low, moderate or highly resistant to the acaricide according to the decision tree shown in table 2. Abbott's formula can be used to correct for a mortality of 10% or below in the controls. A 90% cut-off is chosen based on the WAAVP guidelines recommendation for acaricide efficacy. If the test result indicates high resistance levels even at lOx the recommended concentration, farmers are advised to move to an alternative acaricidal class.

[0053] Table 2

[0054] Although the invention has been explained in further detail above on the basis of merely a single embodiment, it should be clear that the invention is by no means limited to this. On the contrary, many variations and manifestations are still possible within the scope of the invention for a person skilled in the art. Particularly, the bioassay device and method according to the invention may likewise be applied to a broader range of ixodid ticks than the ones mentioned hereinbefore.

Claims

Claims:

1. A bioassay device for determining acaricide resistance by ticks, particularly ixodidae, comprising at least one multi-cell structure for containing one or more sample ticks, comprising a first cell and a group of further cells, said cells being mutually isolated from one another, wherein said first cell is void of said acaricide, and wherein said further cells comprise said acaricide to be tested in increasing concentrations, characterized in that said first cell is empty or comprises a first impregnated piece of absorbing tissue , in that said further cells each comprise a further impregnated piece of absorbing tissue, in that said first impregnated piece of absorbing tissue is impregnated with a diluent only, and in that said further impregnated pieces of absorbing tissue comprise said diluent with said increasing concentrations of an acaricide to be tested.

2. The bioassay device according to claim 2, characterized in that said at least one multi-cell structure comprises at least one further multi-cell structure comprising a further group of further cells containing further impregnated pieces of absorbing tissue comprising said diluent with increasing concentrations of a further, different acaricide to be tested.

3. A bioassay device according to claim 1 or 2, characterized in that said diluent comprises an oily liquid, particularly olive oil.

4. The bioassay device according to claim 1, 2 or 3, characterized in that said further cells comprise a recommended concentration of the acaricide to be tested and respective multitudes of said recommended concentration.

5. The bioassay device according to claim 4, characterized in that said multitudes of said recommended concentration comprise five times said recommended concentration and ten times said recommended concentration.

6. The bioassay device according to anyone of the preceding claims, characterized in that the cells of said multi-cell structure are arranged in at least one row holding multiple positions, said first cell occupying a first position in said row, and in that said group of further cells is distributed over further positions in said row in an order of increasing acaricide concentration.

7. The bioassay device according to claim 5, characterized in that said row is a first row of a number of substantially identical rows for testing the acaricide resistance of said acaricide.

8. The bioassay device according to claim 5 or 6, characterized in that said multiple-cell structure comprises three substantially identical rows of cells for each acaricide resistance to be determined.

9. The bioassay device according to anyone of the preceding claims, characterized in that said multi-cell structure is provided by a container having several pockets, said pockets corresponding to the cells of said multi-cell structure respectively.

10. The bioassay device according to claim 9, characterized in that said pockets are arranged in rows, and in that said container comprises a distinct row of empty pockets.

11. The bioassay device according to claim 10, characterized in that said cells are provided by corresponding rows of pockets of said container, in that said container comprises a first group of mutually adjacent rows of pockets for testing said first acaricide, in that said container comprises a further group of mutually adjacent rows of pockets for testing a further, different acaricide, and in that said first groups of rows is separated from said further group of rows by said distinct row of empty pockets.

12. The bioassay device according to anyone of claims 9 to 11, characterized in that at least said further cells of said at least one multi-cell structure are provided by pockets containing said impregnated pieces of said absorbing tissue, said impregnated pieces ofabsorbing tissue comprising pieces of fabric or non-woven material being arranged inside said pockets.

13. The bioassay device according to claim 12, characterized in that said pockets are confined by portion of a bottom of said container and by closed walls that separate said pockets , and in that said impregnated pieces of tissue occupy an majority of said portion of said bottom within a pocket and extend over at least part of a height of said walls.

14. The bioassay device according to claim 12 or 13, characterized in that said tissue comprises nylon.

15. The bioassay device according to anyone of claims 9 to 14, characterized in that each pocket is sealable by an individual transparent lid and in that said container comprises a transparent further lid jointly securing the individual lids of the pockets of the container in a closed position.

16. The bioassay device according to claim 15, characterized in that said container, including said lid, is formed from transparent polypropylene.

17. The bioassay device according to anyone of claims 9 to 16, characterized in that said pockets are labelled according to their acaricide concentration.

18. The bioassay device according to anyone of the preceding claims, characterized in that said acaricide comprises at least one acaricide from a group containing pyrethroids, formamidines, organophosphates, macrocyclic lactones, phenylpyrazoles, and isoxazolines.

19. The bioassay device according to anyone of the preceding claims, characterized in that said diluent comprises a mixture of trichloro-ethylene or acetone and vegetable oil, particularly olive oil.

20. The bioassay device according to claim 19, characterized in that said mixture comprises trichloro-ethylene and said vegetable oil in a ratio of at least substantially 2:1.

21. A method of using the bioassay device according to anyone of the preceding claims, characterized in that equal numbers of semi-engorged ticks are distributed over the cells of said at least one multi-cell structure, particularly an equal number of five to eight ticks in each cell, and a mortality per cell is determined after dark exposure of said ticks at room temperature under moist conditions.

22. The method according to claim 21, characterized in that said bioassay device is loaded with at least one acaricide comprising an active compound from a group containing containing pyrethroids, organophosphates, macrocyclic lactones, phenylpyrazoles and isoxazolines, and kept in a closed condition in a sealed enclosure, particularly a zipper bag, containing moisturizing means, particularly a moist paper towel, for 24 hours.

23. The method according to claim 21, characterized in that said bioassay device is loaded with at least one acaricide comprising an active compound from a group containing formamidines, and kept in a closed condition in a sealed enclosure, particularly a zipper bag, containing moisturizing means, particularly a moist paper towel, for 96 hours.

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