Biossay of acaricide resistance on three common cattle tick species at Holotta, Central Ethiopia

ARTICLE ORIGINAL Biossay of acaricide resistance on three common cattle tick species at Holotta, Central Ethiopia J. YILMA*, G. ADAMU and E. ZERBINI Department of Pathology and Parasitology, Faculty of Veterinary Medicine, Addis Ababa University, P.O. Box 34, Debre Zeit, Ethiopia Ministry of Agriculture, Addis Ababa International Livestock Research Institute, Addis Ababa SUMMARY Bioassay of acaricide resistance on three common cattle ticks was carried out on four farms in Holotta area, Central Ethiopia, using a standardized FAO Acaricide Resistance Test Methods. Larval progeny of Boophilus decoloratus, Rhipicephalus e. evertsi and Amblyomma variegatum were subjected to larval packet test (LPT) employing four different acaricides (Dieldrine, Diazinon, Chlorfenvinphos and Coumaphos) each with five concentration levels. Strains of Boophilus decoloratus, Rhipicephalus e. evertsi, originated from an area where there is no tradition of acaricide application for tick control, were used as reference ticks. Extremely high susceptibility of reference tick strains was confirmed to all the tested acaricides. Statistical analysis of pooled mean mortality rates revealed that B. decoloratus strains were found resistant to Dieldrine and Diazinon in all studied farms ; and to Chlorfenvinphos and Coumaphos only at the Sadamo farm. In addition, strains of R. e. evertsi from the research station farm revealed a slight degree of resistance to Coumaphos. A. variegatum strains from all farms showed high degree of susceptibility to all tested acaricides. The finding of organophosphate resistance is the first report of its kind in Ethiopia. Extensive uses of acaricides like BHC and Bacdip; irregular spraying, failure to maintain adequate lethal concentrations and other managerial constrains hampering the successful use of acaricides are anticipated causes of the emerging acaricide resistance in the study area. Pertinent recommendations were made to alleviate the existing problem in the study area as well as in other affected regions in the country. RÉSUMÉ Étude de la résistance aux acaricides chez trois espèces de tiques très répandues en Éthiopie Centrale, à Holotta. Par J. YILMA, G. ADAMU et E. ZERBINI. Une étude de la résistance aux acaricides a été menée sur trois types de tiques communes chez le bétail dans quatre fermes d Ethiopie Centrale (région d Holotta). Cette étude a utilisé la méthode standardisée par la FAO pour l étude de la résistance aux acaricides. Des larves de Boophilus decoloratus, Rhipicephalus e. evertsi et Amblyomma variegatum ont été traitées par le «Larval Packet test» (LPT) avec quatre acaricides différents : Dieldrine, Diazinon, Chlorfenvinphos et Coumaphos, chacun administré à cinq concentrations différentes. B. decoloratus se révèle résistant au Dieldrine et au Diazinon dans toutes les fermes étudiées et résistant au Chlorfenvinphos et au Coumaphos uniquement dans la ferme Sadamo. R. e. evertsi montre une faible résistance au Coumaphos. A. variegatum, dans toutes les fermes, résiste à un haut degré à tous les acaricides. Le mauvais emploi des acaricides est responsable de l apparition de ces résistances. Des recommandations pertinentes sont données à ce sujet. KEY-WORDS : bioassay - acaricide resistance - cattle - B. decoloratus - R. e. evertsi - A. variegatum - Holotta - Ethiopia. MOTS-CLÉS : résistance aux acaricides - B. decoloratus - R. e. evertsi - A. variegatum - Holotta - Ethiopie. Introduction Ticks are considered as the most damaging livestock pests on a global scale. Ticks are responsible for a great diversity of livestock health problems. Approximately 80 % of the world s cattle population are at risk to tick infestation and tick-borne diseases [11]. Their infestation alone can give rise * To whom all correspondences and offprint requests should be addressed ; Fax : 251-1-33 99 33 ; e.mail : vet.medicine@telecom.net.et to severe irritation and trauma, which results in substantial economic losses from reduced milk yield, skin and hide damage and reduced weight gain. As vectors of pathogens, ticks are second only to mosquitoes and are responsible for the transmission of protozoal, riskettsial, bacterial and viral deseases among domestic animals [6]. According to McCOSKER [8], it is estimated that the global costs of tick control and productivity losses account for some 700 million USD annually. Comparative data on the economic losses from ticks and tick-borne disease in the African livestock are lacking.

386 YILMA (J.) AND COLLABORATORS However, an estimated 168 million USD in Eastern, Central and Southern Africa is caused only by East Coast Fever [2]. The impacts from such losses are exacerbated in tropical and sub-tropical environments where the host is subject to additional stresses associated with sub-optimal nutrition and high environmental temperature. In Ethiopia, an annual conservative estimated loss of 1 million USD is attributed to the downgrading of hides and skin due to tick infestation [19]. With the inclusion of losses from reduced productivity, deaths and costs of tick control, the estimated total loss will obviously be much greater than this. Application of acaricides in dips and sprays remains as the conventional means of tick control over the past many decades. This method has resulted in a profound influence on livestock productivity through dramatic reduction in the prevalence of tick infestation and tick-borne diseases. Acaricides, particularly the stable and persistent ones, have provided rapid and efficient means of combating livestock ticks in many parts of the world [15]. This method has been and still practiced in Ethiopia and likely to remain as one of the most dependable weapon in the foreseeable future [12]. However, their injudicious use represents the greatest threat to livestock industry of many countries. Over-reliance and misuse of most chemical acaricides have resulted in a rapid emergence of resistant tick populations and objectionable pesticide residues in the environment. In Ethiopia, unconfirmed reports from different corners of the country strongly suggest the emerging presence of tickacaricide-resistance. The situation is further compounded by the absence of an effective legislation for acaricide importation, marketing and monitoring. Information on the status and magnitude of acaricide resistance is of paramount importance in deciding the appropriate tick and tick-borne disease control strategy in different localities in the country. The present bioassay study is, thus, designed to assess the presence and magnitude of acaricide resistance in three common cattle tick species at Holotta area, Central Ethiopia. Material and methods THE STUDY AREA AND FARMING The study was conducted in four farms at Holotta area, Central Ethiopia, located 38 3 E, 9 N, at an altitude of 2400 m asl. The area receives a mean annual rainfall of 1200 mm, 70 % of which comes from the big rains that run from June through September. The average minimum and maximum temperature ranges are 2-9 C and 20-27 C, respectively. Farming is generally dominated by smallholder crop-livestock mixed system in which the cattle husbandry mainly stands for milk production and traction. The predominant cattle breeds are zebu with some European crosses. Cattle are grazed during daytime and supplemented with crop residues and hay in the evenings. Tick infestation is one of the major health constraints in the area. Control measures heavily relay on chemical means (usually employing BHC, Asuntol, Bacdip and Supona) applied by hand spray or spray races. The practice of acaricide application is irregular, often carried out when engorged adult ticks are apparent in large numbers on cattle. ANIMALS A total of 80 cross-bred (Boran x Friesian) cattle from four farms (Jerbasefer, Chiri, Sadamo and Research Station) were randomly selected for the purpose of tick collection. No particular consideration was given to sex and age of animals. TEST AND REFERENCE TICKS Fully engorged females of Boophilus decoloratus, Rhipicephalus e. evertsi and Amblyomma variegatum were collected from the indicated four farms at Holatta and identified using standard keys set by MOREL [9]. For oviposition and harvest of sufficient larval progenies, five ticks from each species were placed in separate glass tubes covered with gauze and incubated at a temperature of 27 ± 1 C, and a relative humidity of 85-90 %, as per standard procedures recommended by FAO [4]. Strains of Boophilus decoloratus and Rhipicephalus e. evertsi, collected from Wollenchiti area (East Shewa zone) where the usage of acaricides was confirmed to be minimum or nil, have served as reference of susceptible population. THE TEST METHOD FAO-Acaricide resistant test kit, containing paper impregnated with five concentration levels of Dieldrin, Diazinon, Chlorfenvinphos, and Coumaphos (Table I) obtained from WARRC, Berlin, Germany, were employed. The test was conducted according to the larval packet test (LPT) described by STONE and HAYDOCK [14], that allows assessing the presence of acaricide resistance. The procedure was applied to all tick species and repeated over several occasions based on the number of samples and sampling frequency. The LPT database obtained from the reference ticks provided tangible comparison to that of the test ticks. DATA COLLECTION AND ANALYSIS The test packets were opened after 24 hours of incubation, laid flat on polystyrene block and larvae counted by using tally counter and a magnifying lens. Only larvae capable of moving were regarded as alive. All other larvae, including those that move appendages but do not walk were regarded as dead. In order to find out whether the mortality was due to the acaricide and not due to other causes, the control mortality was always taken into consideration. Data were then computed using the following formulas : Mortality (%) = Dead Tick larvae count x 100 Total Tick larvae count In tests with control mortality between 0-5 %, every mean mortality was corrected using Abott s formula : Corrected mortality (%) = (% test mortality - % control mortality) x 100 100 - % control mortality

BIOASSAY OF ACARICIDE RESISTANCE ON THREE COMMON CATTLE TICK SPECIES AT HOLOTTA, CENTRAL ETHIOPIA 387 Correction is not necessary if the control mortality is 0 % and the test mortality is 100 %. Tests with control mortality over 5 % were rejected. Pooled mean mortality (%) = Mortality i + Mortality ii... + Mortality n N Where i, ii,... n denote the 1 st, 2nd,... and the n th sample data, respectively, and N stands for the total sample size. STATISTICAL ANALYSIS Obtained data were analyzed by Chi-square (χ 2 ) statistics and the susceptibility differences between tick species from the four study farms exposed to different acaricide concentration levels were determined. Ninety-five percent probability level (P < 0.05) was considered significant. Results REFERENCE TICKS Both B. decoloratus and R. e. evertsi used as reference strains revealed highest mortality up on exposure to different concentrations of acaricides tested. The efficacy of Dieldrine, Diazinon, Chlorfenvinphos and Coumaphos, against both tick species, were in the order of 99.75 %, 99.35 %, 100 % and 99.73 %. The overall mortality for B. decoloratus and R. e. evertsi were 99.58 % and 99.88 %, respectively (Table II). TEST TICKS B. decoloratus The pooled mean mortality rates for B. decoloratus exposed to different concentration levels of Dieldrine, Diazinon, Chlorfenvinphos, and Coumaphos were 27 ± 6.2, 55.02 ± * Dieldrine is a reliable Taxaphane and Lindane (BHC) resistant indicator Diazinon is considered as a general indicator of Organophsphate resistance TABLE I. Types of acaricides and the respective concentration levels contained in the FAO-Acaricide Resistance Test Kit. TABLE II. Pooled mean percentage mortality rate in reference tick strains exposed to different concentration levels of acaricides.

388 YILMA (J.) AND COLLABORATORS 8.61, 94.17 ± 3.19 and 94.7 ± 2.64, respectively. At highest concentration levels, Dieldrine and Diazinon revealed mean tickicidal effects of 39.58 ± 9.07 and 81.05 ± 8.65, respectively in contrast to 99.88 ± 0.13 and 100 of Chlorfenvinphos and Coumaphos (Table III). The least mortality rates (16.22 ± 1.68 and 37.08 ± 7.52) of Dieldrine and Diazinon were recorded on strains of B. decoloratus from Jerbasefer and Research Station farms, respectively. The minimum pooled mean mortality rate recorded for Chlorfenvinphos and Coumaphos were 85.34 ± 5.08 and 87.20 ± 5.89, respectively, and both occurred on ticks from the Sadamo farm (Table IV). The killing efficacy of Dieldrine and Diazinon to B. decoloratus from all farms and at all concentration levels was significantly inferior to that of Chlorfenvinphos and Coumaphos (p < 0.001). R. e. evertsi Overall high-pooled mean mortality rates for the tested acaricides exceeded 90 %, the minimum and maximum TABLE III. Pooled mean percentage mortality of test ticks (B. decoloratus, R. e. evertsi and A. variegatum) exposed to different concentration levels of acaricides. TABLE IV. Pooled percentage mortality of test ticks (B. decoloratus, R. e. evertsi and A. variegatum) from different study sites at Holotta, Central Ethiopia.

BIOASSAY OF ACARICIDE RESISTANCE ON THREE COMMON CATTLE TICK SPECIES AT HOLOTTA, CENTRAL ETHIOPIA 389 values being 91.81 ± 1.88 for Coumaphos and 98.84 ± 0.61 for Dieldrine, respectively. For all tested acaricides except Coumaphos, the killing efficacy reached 100 % at or above the 3 rd and 4 th concentration levels (Table III). Similarly, high degree of susceptibility of R. e. evertsi strains was observed in all study farms (Table IV). There was no statistically discernable difference (p > 0.05) in susceptibility of R. e. evertsi up on exposure to various concentration levels and between studied farms. A. variegatum The global pooled mean mortality rates of A. variegatum were 99.28 ± 1.40, 99.78 ± 0.12, 98.51 ± 0.71 and 97.20 ± 1.48, for Dieldrine, Diazinon, Chlorfenvinphos and Coumaphos, respectively. All acaricides, except Dieldrine, showed 100 % tickicidal effect at and above the 3 rd or 4 th concentration levels (Table III). The efficacy of all tested acaricides against strains of A. variegatum exceeded 90 % in all studied farms, the minimum value obtained being 91.78 ± 3.70 at Chiri farm (Table IV). Statistical comparision of results revealed that there were no significant differences (p > 0.05) between mortality rates of A. variegatum strains from different farms and exposed to different acaricides at different concentration levels. An overall assessment of results from all farms showed that the susceptibility of B. decoloratus to Dieldrine and Diazinon were significantly lower (p < 0.001) than R. e. evertsi and A. variegatum. Conversely, no significant variation (p > 0.05) was detected between species of test ticks exposed to different concentration levels of Chlorfenvinphos and Coumaphos. Discussion Resistance to pesticides is a genetically controlled phenomenon, believed to develop largely as a result of selection of pre-adaptive mutants for detoxification, target site insensitivity, or other means of survival in the presence of insecticides. Acaricide resistance is nature s response to widespread use of long lasting chemicals whose presence constitute a selective influence on genetically endowed survivors [1]. In Africa, resistance could ultimately lead to a much greater dislocation of the cattle industry than elsewhere because of the existence of more serious problems associated with threehost-ticks such as Rhipicephalus, Amblyomma and Hyalomma species [16]. Resistance to acaricide is usually recognized because of failure to obtain a satisfactory kill of the parasitic stages on treated animal. The first tick resistance outbreak was reported from Australia on cattle tick, Boophilus microplus, to arsenic compounds and subsequently from South Africa and Latin America. Resistance to this chemical did not appear until after several decades of use, but resistance to chlorinated hydrocarbons appeared with far greater rapidity and is now complicated by cross-resistance between various compounds of the group [10]. In the present study, B. decoloratus strains from all farms showed high degree of resistance to Dieldrine and Diazinon, and to Chlorfenvinphos and Coumaphos at the Sadamo farm. YEHUALASHET and GEBREAB [18] reported that most B. decoloratus from western Ethiopia were resistant to organochlorine compounds (Toxaphane). REGASSA and DE CASTRO [12] have also reported the presence of Toxaphaneresistance in B. decoloratus from the same region. Possibly because of its one-host behaviour and its prolonged exposure to treatments, the genus Boophilus has shown the greatest tendency to develop resistance [3]. The finding of Dieldrine resistance in B. decoloratus strain in the present study may be associated with prolonged application of BHC and improper application techniques characterized by frequent mechanical defects resulting in failure to maintain adequate concentrations. Resistance to chlorinated hydrocarbons is usually complicated by cross-resistance between various compounds in the group. WHITNALL, et al., [17] demonstrated that BHCresistant strains of B. decoloratus in South Africa were also resistant to Toxaphane. HITCHCOK [5] found, in laboratory trials that engorged ticks of BHC-Resistant B. microplus from Queensland, Australia, were also resistant to Toxaphane. It is, therefore, highly likely that B. decoloratus strains from Holotta area may also be resistant to Toxaphane. B. decoloratus populations from all studied farms, in the present study, have also developed high level of resistance to Diazinon. The relatively higher level of resistance to this chemical detected at the Research Station farm is due probably to over reliance and intensive utilization of Bacdip over the past few years. This is the first report of its kind on the occurrence of organophosphorus resistance in Ethiopia. However, resistance of B. decoloratus to organophosphorus acaricide has been recorded in South Africa [13] and Zambia [7], Chlorfenvinphos and Coumaphos, on the other hand, revealed high degree of tickicidal efficacy to B. decoloratus strains from all but Sadamo farm. However, it is generally believed that both acaricides are potent if applied at the correct concentration and interval of time and can be considered as acaricides of choice for the control of ticks and tick-borne diseases in the area. R. e. evertsi test ticks did not reveal any resistance to both organochlorine and organophosphates in three of the studied farms, and only slight degree of resistance was noted on strains from the Research Station farm. Previous works of YEHUALASHET and GEBREAB [18], REGASSA and DE CASTRO [12] in Western Ethiopia indicated the existence of low level resistance of this tick species to organochlorine compounds. It was showed, however, that R. e. evertsi developed resistance to BHC and Toxaphane in several African regions, particularly in Eastern Africa, where control of theileriosis necessitated intensive application of these chemicals [16]. In this study, all strains of A. variegatum were found highly susceptible to all tested acaricides. Similarly, there is no published report on the occurrence of acaricide resistance of this tick species elsewhere in Ethiopia. In Africa, varying degrees of resistance to Toxaphane and BHC were documented in Zambia and Tanzania [16].

390 YILMA (J.) AND COLLABORATORS The tick control scenario in the study area is featured by several managerial constraints such as irregular spraying and improper mixing of chemicals, which may lead to failure to maintain the adequate lethal concentration. It is, thus, recommended to revise the existing methods and consider other tick control strategies that could best address the emerging acaricide resistance problem in the study area. Problems posed by ticks and their diversified interaction with the ecosystem are also too dynamic to be solved by reliance on one component of the control systems [20]. The necessity to develop a sustainable and integrated scheme, in which the isolated methods may be individually inadequate but together with others may exert a vigorous synergy, is justified. This method consists of the use of tick-resistant cattle breeds, pasture management, habitat modification and immunization in combination with rational use of acaricides. Although difficult to materialize in developing countries like Ethiopia, the prospect of this approach is considerable, as it is the most sustainable method and keeps enzootic stability undisturbed. Appropriate and rational use of any new replacement acaricide for the resistant affected chemicals should be, in principle, encouraged. However, appropriate policies must be set for importation, assessment of efficacy and monitoring of their use in the field. In addition, strict quarantine should be drawn to reduce the possibility of introduction of ticks and tick-borne diseases with imported cattle. Surveys on acaricide resistance need to be carried out in major livestock production areas in the country in order to gather a comprehensive database on the magnitude of the problem at a national scale. Acknowledgements Acaricide Resistant Test Kits were generously provided by FAO-World Acaricide Resistance Reference Center (WARRC), Berlin, Germany. 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