Exp Appl Acarol (2014) 63:413–422 DOI 10.1007/s10493-014-9789-8
Determination of discriminating dose and evaluation of amitraz resistance status in different field isolates of Rhipicephalus (Boophilus) microplus in India Sachin Kumar • Anil Kumar Sharma • D. D. Ray • Srikant Ghosh
Received: 20 August 2013 / Accepted: 5 March 2014 / Published online: 22 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Field tick isolates of Rhipicephalus (Boophilus) microplus were collected from eleven districts located in the northern and eastern states of India to access the resistance status to ‘‘Amitraz’’. Adult immersion test was optimized using laboratory reared acaricide susceptible IVRI-I line and minimum effective concentration was determined as 487.7 ppm with 95 % confidence interval of 455.8–521.8. The discriminating concentration was determined as 975.4 ppm and was tested on female ticks collected by two stage stratified sampling from organized dairy farms and villages. Based on three variables, viz., mortality, egg masses and reproductive index, the resistance level was categorized. Resistance to amitraz was detected at level I in 3 isolates (RF = 1.56–5.0), at level II in 6 isolates (RF = 9.3–23.3) and at level III in 1 isolate (RF = 27.3) whereas one isolate was found susceptible. The highest resistance was found in the SKR isolate (RF = 27.3) and minimal resistance was detected in the N-24P isolate (RF = 1.56). These experimental data will help in designing tick control strategy which is suffering from acaricide failure and to overcome development of resistance in ticks. Keywords Amitraz Discriminating concentration Rhipicephalus (Boophilus) microplus Resistance
Introduction India is blessed with a bovine population of 199.10 million cattle and 105.30 million buffalo, accounting for 16.2 and 56.9 %, respectively, of world bovine populations and stands first in the world in size of bovine population (Annual Report 2011–12). Almost all
S. Kumar A. K. Sharma D. D. Ray S. Ghosh (&) Entomology Laboratory, Parasitology Division, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, UP, India e-mail: [email protected]
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of these animals are suffering from tick infestations at different times of the year. Amongst 106 valid tick species reported from India, Rhipicephalus (Boophilus) microplus is widely prevalent and is the most damaging species infesting livestock, zoo and wild animals (Ghosh et al. 2007). Economic losses to cattle producers from ticks and tick-borne diseases (TTBDS) has been estimated at US $ 13–14 milliard globally on an annual basis (de Castro 1997), to which Brazil and Australia contribute US $ 2 milliard (Grisi et al. 2002) and Aus $ 175 million (Playford et al. 2005), respectively. In India, the control cost of TTBDS in the dairy sector has been estimated at $498.7 million per annum (Minjauw and Mc Leod 2003). This tick species causes severe economic losses through anorexia, toxicosis, decreased milk yield, meat production, damaged hides and by transmission of diseasecausing agents, babesiosis and anaplasmosis (Rodrı´guez-Vivas et al. 2004; Ghosh et al. 2007). The use of different groups of acaricides viz. organophosphates (OP), synthetic pyrethroids (SP), amidines and macrocyclic lactones is the most common tick control method. However, due to continuous and indiscriminate use of insecticides, resistance has been detected in R. (B.) microplus almost every chemical that is registered for use against it (Whalon et al. 2008). In India, although cattle owners have reported inefficiency of these acaricides in field conditions, only limited data on resistance to these chemicals are currently available (Kumar et al. 2011; Vatsya and Yadav 2011; Sharma et al. 2012a; Shyma et al. 2012). Amitraz is a triazapentadiene compound, a member of the amidine chemical family introduced in 1972. It acts as a non-systemic octopaminergic agonist and has been used for the control of ticks on cattle (Garris and George 1985; Curtis 1985; Kagaruki 1996), dogs and wild animals (Pound et al. 2000; Elfassy et al. 2001; Kumar et al. 2001), parasitic mites of honey bee (Floris et al. 2001), and other ectoparasites of livestock (Curtis 1985). It was postulated that formamidine pesticides exert their toxic effect on target species by interaction with octapamine receptors of the CNS (Dudai et al. 1987), and possibly also by inhibition of monoamine oxidases (Atkinson et al. 1974; Schuntner and Thompson 1976). In India, amitraz is one of the commonly used acaricides for the control of cattle and dog ticks (Mathivathani et al. 2011) but concerted efforts have not been directed towards monitoring of resistance against the chemical acaricide. Amitraz resistance was detected in the Alfonso strain of Mexico (Soberanes et al. 2002), the Uma & Ultino strain of Australia (Kunz and Kemp 1994), and the R. (B.) microplus population of Columbia (Benavides et al. 2000), South Africa (Strydom and Peter 1999) and Brazil (Furlong 1999; Miller et al. 2002). However, except for a limited study by Singh et al. (2013), a survey has never been conducted to monitor susceptibility/resistance patterns of different Indian isolates of R. (B.) microplus against amitraz.
Materials and methods Acaricide Technical grade amitraz (99 %) was procured from AccuStandardÒ, USA, and was used for preparing a 49,500 ppm stock solution by dissolving 100 mg in 2 ml methanol. For the experimental bioassay, different aqueous concentrations of the acaricide were prepared (v/v) from the stock solutions and tested against the susceptible IVRI-I line (national registration no. NBAII-IVRI-BM-1-1998) and field isolates of R. (B.) microplus.
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Animals Weaned cross-bred (Bos taurus male 9 B. indicus female) male calves were reared in tick proof animal houses of the division of Parasitology, Indian Veterinary Research Institute and fed with calf starter, milk, concentrate mixture, wheat brans and water ad lib. Separate batches of five to six calves were used to maintain the reference susceptible IVRI-I line and field collected ticks. The calves were reared according to the guidelines of the ‘‘Committee for the Purpose of Control and Supervision on Experimentation on Animals’’, a statutory Indian body. Reference tick line The susceptible reference IVRI-I line was used as the standard to assess susceptibility/ resistance status in collected field isolates. The colony has been maintained in the Entomology Laboratory of Indian Veterinary Research Institute for the last 15 years and has not been exposed to any acaricides. The susceptibility status of the colony was monitored by periodical testing against several organo-phosphates, organo-chlorines, SP and formamidine compounds in independent bioassays. The homogeneity amongst different generations of IVRI-I line was established by uniform entomological parameters and by analyzing the sequences of 16s rDNA gene (Accession Nos. GU222462, GU323287, GU323288 (Kumar Rinesh et al. 2011). Determination of DC, LC50 and LC95 values The discriminating concentration (DC) of the test acaricide was determined performing dose-response bioassays with the IVRI-I line adopting the Adult immersion test (AIT) as mentioned by Drummond et al. (1973) and Benavides et al. (1999) with minor modifications. A serial of six concentrations of amitraz (49.5, 148.5, 247.5, 297.0, 396.0, 495.0 ppm) were prepared in distilled water from a stock solution. The engorged females of the IVRI-I line were thoroughly cleaned, dried on tissue paper and kept in Petri dishes padded with filter paper. Ticks were weighed and each replication was kept in different Petri dishes with proper markings for in vitro experiments. The pre-weighed engorged females were dipped in different concentrations of the test acaricide for 2 min and then dried in filter paper before transferring into the Petri dishes. After 24 h, ticks were transferred to the glass vials covered with cotton cloth and were kept in a precision (B.O.D.) incubator maintained at optimum conditions of 28°C and 85 ± 5 % RH. The biological parameters were monitored upto 14 days and the ticks which did not oviposit were considered as dead. The control groups were treated in similar manner in 5 % methanol as the solvent content in different concentrations of amitraz was equal to or less than 5 % level. Earlier, the toxicity of methanol against the IVRI-I line was studied and no adverse effect of dipping of ticks for 2 min in 50 % methanol was observed upto 14 days (Sharma et al. 2012b). The mortality of ticks was recorded regularly by observing the loss of motility and pedal reflex after exposing to light. The data on mortality, egg masses, RI and inhibition of oviposition (IO) were collected. The following parameters were compared: (1)
Mortality: Data were recorded upto 14 days post treatment (dpt) when normal ticks complete egg laying.
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(2) (3) (4)
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The egg masses laid by the live ticks were recorded. RI = egg weight (EW)/ engorged female weight (IFW) treated 100 % Inhibition of oviposition ðIO % Þ ¼ RI control-RI RI control
Dose response data were analyzed by probit method (Finney 1962) using GraphPad Prism version 4.0, San Diego, CA, USA. The LC50 and LC95 values of amitraz were determined applying regression equation analysis to the probit transformed data of mortality. The differences in mean values of entomological data among the groups were analyzed by one way ANOVA. The DC was determined as 2 9 LC95 against the reference IVRI-I line (Jonsson et al. 2007). Sampling and characterization of field isolates A two stage stratified sampling method was adopted to collect samples from organized dairy farms and from villages of different districts located in the states of Uttar Pradesh, Bihar, Punjab, Rajasthan and West Bengal. Both cattle and buffalo populations were screened for tick infestations. A questionnaire was developed to collect basic information about the mode, frequency, type and dose of application of acaricides, and owners experience on efficacy of the commonly used acaricides. The following isolates : RBL, BEG, DRB, MKT, COR, BHT, SKR, JPR, ALW, NAD and N-24P were collected from both organized and unorganized farms. The areas of collection were upper Gangetic plains region situated at 25°N–26°N, 100°E–81°E (Uttar Pradesh), middle Gangetic plains region situated at 25.15°N–25.45°N, 85.4°E–86.3°E (Bihar), trans-Gangetic plains region situated at 30°N–29°N, 74°E–74°E (Punjab), western dry region situated at 23°N–25°N, 77.4°E– 78°E and central plateau and hill region situated at 25°N–27.6°N, 73°E–75.1°E (Rajasthan), the lower Gangetic plains situated at 22°N–24°N, 88°E–89°E (West Bengal). In the upper, lower and middle Gangetic plain regions the annual rainfall ranges from 900–1,500 mm with temperatures of 4–45 °C and 80–90 % humidity. In the trans-Gangetic plains, the annual rainfall ranges from 200–380 mm and the temperature from 12.8–33.9 °C with 79–85 % humidity. The western dry, central plateau and hill regions experience annual rainfall in the range of 200–650 mm with temperatures ranged from 5–45 °C and 47–74 % humidity. Engorged females of all the isolates were collected from animal sheds in separate vials, covered with cotton cloths to allow air and moisture exchange, and were transported to the laboratory. All the samples collected from a particular area were mixed and washed in water, labeled and kept at 28°C and 85 ± 5 % relative humidity. The AIT was conducted at the spot where engorged ticks were collected in a large number. The ticks were kept for laying eggs where few ticks were collected. In the laboratory, the egg masses of different engorged females collected from a particular area were pooled and pooled larvae were released on calves for feeding. After 18–20 days of feeding the engorged females were collected from the animal shed and resistance/susceptibility to amitraz was determined by AIT using statistically significant number of ticks. Resistance factors (RF) for field tick isolates were worked out by dividing LC50 of field ticks using different grades of DC i.e., 29, 49, 69 by LC50 of IVRI-I line (Castro-Janer et al. 2009). On the basis of RF, the resistance level in the field population of R. (B.) microplus was classified as susceptible (RF B 1.4), level I resistance (RF = 1.5–5.0), level II resistance (RF = 5.1–25.0), level III resistance (RF = 26–40) and level IV resistance (RF C 41) (Kumar et al. 2011).
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Results Determination of LC50 and LC95 values A direct relationship of the dose-response mortality of the IVRI-I line of R. (B.) microplus treated with different concentrations of amitraz was recorded (Fig. 1). A 100 % mortality of ticks with complete IO was recorded when treated with 495.0 ppm concentration. The Log probit model indicated a high slope value of 3.49 ± 0.46 revealing high drug response and 94 % correlation factor. However, negative slopes of egg masses (-19.97 ± 18.1) and RI (-0.22 ± 0.13) showed that both the parameters were adversely affected by the acaricide. Analyzing the log-probit equation, the LC50 was calculated as 165.0 ppm with a 95% confidence limit of 155.7–174.9 while the LC95 value was 487.7 ppm with a 95 % confidence limit of 455.8–521.8. The DC was calculated to be 975.4 ppm (Table 1). Survey areas In most of the areas surveyed, the tick activity was high from April to October and then generally declined to a minimum during winter. At a few places the farmer reported tick activity on their animals round the year. According to the veterinary records it was noted that formamidine, OP, SP and macrocyclic lactones were frequently used for tick control in both organized and unorganized farms. The farmers are using acaricides only when they see ticks on their animals. The use of acaricides in organized dairy farms was twice or thrice a month. The most common methods of acaricide application were spray, injectable route and swabbing. It was noticed that no regular acaricide treatment regime was maintained in any of the surveyed areas irrespective of the type of farms. Besides application of acaricides, natural control by birds was also seen in unorganized farms where animals were allowed for grazing in the open fields. At most of the places, the women were involved in hand picking of ticks from their animals in leisure hours.
Fig. 1 Dose-dependent mortality and inhibition of oviposition (IO) of Rhipicephalus (B.) microplus IVRI-I line treated with amitraz
Table 1 LC50 and LC95 values of amitraz obtained by adult immersion test (AIT) for the susceptible IVRI-I line of Rhipicephalus (B.) microplus Variables
Slope ± SE
R2
LC50 (ppm) (95 % CL)
LC95 (ppm) (95 % CL)
165.0 (155.7–174.9)
487.7 (455.8–521.8)
Mortality
3.49 ± 0.46
0.94
Egg mass
-19.97 ± 18.1
0.23
-0.22 ± 0.13
0.44
0.14 ± 0.04
0.70
RI % IO
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Table 2 Amitraz resistance status in field isolates of Rhipicephalus (B.) microplus Level of resistancea
Tick isolates
Slope ± SE
LC50 value (95 % CL)
Resistance factor
RBL
3.96 ± 4.32
1917.4 (1826.1–2013.2)
11.6
II
BEG
3.09 ± 1.24
825.1 (771.1–882.8)
5.0
I
DRB
6.53 ± 0.50
1815.7 (1757.8–1864.8)
11.0
II
MKT
5.31 ± 3.78
2066.8 (1983.3–2149.5)
12.5
II
COR
3.34 ± 1.084
1929.1 (1837.2–2025.5)
11.7
II
BHT
1.27 ± 0.16
401.1 (341.2–475.1)
2.4
I
SKR
3.00 ± 1.24
4501.2 (2880.6–3115.7)
27.3
JPR
2.77 ± 1.16
3847.7 (3530.0–4194.0)
23.3
III II
ALW
7.17 ± 4.04
1537.5 (1507.3–1568.2)
9.3
II
NAD
1.08 ± 0.43
99.7 (82.5–120.7)
0.6
Susc.
N-24P
1.53 ± 0.61
255.8 (224.4–291.5)
1.56
I
a
Susceptible = RF \ 1.4, level I = 1.5 \ RF \ 5.0, level II = 5.1 \ RF \ 25, level III = 26 \ RF\ 40
Amitraz resistance pattern The details of LC50 values, RF and level of resistance in different isolates are presented in Table 2. Out of eleven isolates, the maximum LC50 value of 4501.2 with confidence limit of 2880.6–3115.7 was recorded in the SKR isolate collected from large to very large organized animal farms, and frequency of use of different acaricides is very high. Although BHT, SKR, JPR and ALW isolates were collected from comparatively dry areas, the tick problem was at a very high level. The other isolates (DRB, BEG) were collected from comparatively high humid areas and the use of amitraz was practised along with that of pyrethroids at frequent intervals and the resistance factor to amitraz reached upto 11.0. The resistance to amitraz was detected in eleven isolates; resistance at level I in 3 isolates (RF = 1.56–5.0), at level II in 6 isolates (RF = 9.3–23.3) and at level III in one isolate (RF = 27.3) while the NAD isolate was found susceptible (Table 2). The regression analysis of collected tick isolates revealed that the resistant isolates of Level II and III have higher slope values. The highest steep response of amitraz was recorded in DRB isolate showing 99.4 % chance to give the regression line of 6.53 ± 0.50 and isolate came under resistance level II. Similar pattern was recorded in MKT, COR, ALW, RBL and JPR isolates. The SKR isolate with the highest resistance value exhibited 3.00 ± 1.24 slope value with R2 of 0.644 indicating that the drug response was not very high in different DCs on the isolate. The field isolates of resistance level I showed a more homogenous and gradual drug response with steep slopes and higher R2 (Table 2). In case of the reference susceptible IVRI-I line, the dose mortality response was 3.49 ± 0.46 showing a steeper slope with 0.94 R2 value.
Discussion India is the world’s largest producer of milk and dairy farming is considered an important source of income of the farmers’ community. Most dairy herds in northern India are crossbred (Bos taurus 9 B. indicus) cattle and buffaloes. The problem of TTBDS is
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particularly relevant in India because of the favorable environmental conditions for tick survival throughout most parts of the year and the maintenance of susceptible cross-bred animals to improve the production of milk and other animal products. The most widely used method for the control of ticks is the direct application of acaricides to host animals and thus the consumption of insecticides has been increased manifold during the last decades. Amitraz, a formamidine acaricide, plays an important role in the control of the cattle tick, R. (B.) microplus, and other tick species that infest cattle and pet animals. It has minimal toxicity to cattle or humans and no meat withholding period, making it suitable for use in cattle shortly before slaughter (Jonsson and Hope 2007). Although amitraz resistance in R. (B.) microplus was previously reported in several countries, the actual field situation in India has not been worked out due to non-availability of a suitable bioassay technique and the DC required for the detection of resistance. The unavailability of engorged female ticks in sufficient number required to do AIT for the diagnosis of drug resistance is the technical limitation of this bioassay (Jonsson et al. 2007). In response to the lack of a linear response to amitraz in the standard LPT, Thullner et al. (2000) described a dispersal test using a paper grass stem model. This model provided linear responses to amitraz but has not been widely adopted. The larval packet test was further modified by the introduction of formulated commercial acaricide in place of technical product and using a nylon material in place of filter paper (Miller et al. 2002) and (Ducornez et al. 2005). In the Entomology laboratory of IVRI, AIT has been effectively standardized using technical grade insecticides and DCs for deltamethrin, cypermethrin, malathion, diazinon, fipronil, coumaphos and fenvelerate were worked out with repeatability. The technical grade insecticides were selected over commercial formulation because commercial products are prepared with many proprietary ingredients and it is difficult to assess the responses due to active ingredients (Shaw 1966).The response obtained using technical grade insecticides was linear and was effectively used for characterization of ticks collected from six agroclimatic zones of India (Sharma et al. 2012b). Using the same methodology, in the present study, the DC of amitraz was determined effectively as 975.4 ppm using reference IVRI-I lines of R. (B.) microplus. Presently, the working group on parasite resistance recommends AIT (Drummond et al.1973) for the detection of resistance since it is fast, simple and a relatively cheap method to be adopted in the field (FAO 2004). The AIT method has been successfully adopted in Australia (Jonsson et al. 2007), Brazil (Brito et al. 2011) and in India (Kumar et al. 2011; Sharma et al. 2012b;.Kumar Rinesh et al. 2013; Singh et al. 2013) for the detection of resistance to SP, OP, moxidectin and amitraz. The current study reports the resistance status of R. (B.) microplus against amitraz and the mechanism involved needs to be established. The mode of action of amitraz is believed to be interference with nervous system function of the targeted species by binding to the octopamine receptors (Evans and Gee 1980). Several types of octopamine receptors including a putative octopamine- like, G-protein-coupled receptor were identified in insects (Blenau and Baumann 2001), and in R. (B.) microplus (Baxter and Barker 1999), respectively. However, past reports of involvement of metabolic detoxification and mutation of the octopamine receptors were speculated to be the main mechanism of resistance to amitraz (Li et al. 2004). The use of amitraz became more frequent when pyrethroid resistance problems started to hinder the tick control efforts in Mexico. The first case of amitraz resistance was detected in the San Alfonso strain of R. (B.) microplus collected from a ranch in the state of Tabasco (Soberances et al. 2002). Reports of resistance against amitraz are available against R. (B.) microplus ticks from various parts of the world (Jonsson et al. 2000; Li et al.
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2004; Veiga et al. 2012) and the factors associated with increased probability of R. (B.) microplus resistance in Australia were identified as region, frequency, doses and application method (Jonsson et al. 2000). In India, the presence of resistance to different families of chemical acaricides has already been reported by many workers (Sangwan et al. 1993; Kumar et al. 2011; Vatsya and Yadav 2011; Sharma et al.2012b; Shyma et al. 2012; Kumar Rinesh et al. 2013). Recently, Singh et al. (2013) reported gradual development of amitraz resistance in R. (B.) microplus collected from Gujarat state of India. The present study supported the views of Singh et al. (2013) with additional information on development of multi-acaricide resistant tick populations in the areas of collection since all these isolates were found resistant to a number of OP and SP compounds, possible reasons for acaricide failure in many parts of the country. Acknowledgments The authors are grateful to Indian Council of Agricultural Research, New Delhi for funding through World Bank funded National Agricultural Innovation Project No. NAIP/Comp-4/C2066/ 2008-09. Authors are also grateful to the Veterinary officers posted at different tick collection spots for their support.
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Schuntner CA, Thompson PG (1976) Inhibition of a carbaryl oxidising enzyme as the primary lesion in the lethal action of formamidines in Boophilus microplus. J Aus Entomol Soc 15:388 Sharma AK, Kumar S, Tiwari SS, Srivastava S, Kumar Rinesh, Ray DD, Chaudhuri P, Rawat AKS, Ghosh S (2012a) Comparative acaricidal properties of different solvents and surfactants on Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Indian J Anim Sci 82:154–158 Sharma AK, Kumar Rinesh, Kumar S, Nagar G, Singh NK, Rawat SS, Dhakadd ML, Rawat AKS, Ray DD, Ghosh S (2012b) Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus collected from six agro-climatic regions of India. Vet Parasitol 188:337–345 Shaw RD (1966) Culture and organophosphorus resistant strain of B. microplus (Can.) and an assessment of its resistance spectrum. Bull Entomol Res 56:389–405 Shyma KP, Kumar S, Sharma AK, Ray DD, Ghosh S (2012) Acaricide resistance status in Indian isolates of Hyalomma anatolicum. Exp Appl Acarol 58:471–481 Singh NK, Gelot IS, Jyoti Singh V, Rath SS (2013) Detection of amitraz resistance in Rhipicephalus (Boophilus)microplus from North Gujarat, India. J Parasit Dis. doi:10.1007/s12639-013-0280-y Soberanes MJ, Santamaria VM, Fragoso SH, Gracia ZV (2002) First case reported of amitraz resistance in the cattle tick Boohilus microplus in Mexico. Tec Pecu Mex 40:81–92 Strydom T, Peter R (1999) Acaricide and Boophilus spp. resistance in South Africa. In: Fragoso HS, Garcı´a ZV (eds) IV Seminario Internacional de Parasitologı´a Animal. Control de la resistencia en garrapatasy moscas de importancia veterinariay enfermedades que trasmiten, Conasag-Inifarvet-IICaampaveFilasa, Puerto Vallarta, Jalisco, Mexico, pp 35–40 Thullner F, Kemp DH, Mckenna RV, Willadsen P (2000) Dispersal test for the diagnosis of amitraz resistance in tick larvae (Boophilus microplus). In: Kazimirova M, Labuda M, Nuttall PA (eds) Proceedings of the 3rd international conference on ticks and tick-borne pathogens: into the 21st century. Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia, pp 205–208 Vatsya S, Yadav CL (2011) Evaluation of acaricide resistance mechanisms in field populations of Rhipicephalus (Boophilus) microplus collected from India. Int J Acarol 37:405–410 Veiga LP, Souza AP, Bellato V, Sartor AA, Nunes AP, Cardoso HM (2012) Resistance to cypermethrin and amitraz in Rhipicephalus (Boophilus) microplus on the Santa Catarina Plateau, Brazil. Rev Bras Parasitol Vet 21:133–136 Whalon ME, Mota-Sanchez D, Hollingworth RM (2008) Global pesticide resistance in arthropods. CAB Int, 381, Cromwell Press Trowbridge, UK
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Determination of discriminating dose and evaluation of amitraz resistance status in different field isolates of Rhipicephalus (Boophilus) microplus in India Sachin Kumar • Anil Kumar Sharma • D. D. Ray • Srikant Ghosh
Received: 20 August 2013 / Accepted: 5 March 2014 / Published online: 22 March 2014 Ó Springer International Publishing Switzerland 2014
Abstract Field tick isolates of Rhipicephalus (Boophilus) microplus were collected from eleven districts located in the northern and eastern states of India to access the resistance status to ‘‘Amitraz’’. Adult immersion test was optimized using laboratory reared acaricide susceptible IVRI-I line and minimum effective concentration was determined as 487.7 ppm with 95 % confidence interval of 455.8–521.8. The discriminating concentration was determined as 975.4 ppm and was tested on female ticks collected by two stage stratified sampling from organized dairy farms and villages. Based on three variables, viz., mortality, egg masses and reproductive index, the resistance level was categorized. Resistance to amitraz was detected at level I in 3 isolates (RF = 1.56–5.0), at level II in 6 isolates (RF = 9.3–23.3) and at level III in 1 isolate (RF = 27.3) whereas one isolate was found susceptible. The highest resistance was found in the SKR isolate (RF = 27.3) and minimal resistance was detected in the N-24P isolate (RF = 1.56). These experimental data will help in designing tick control strategy which is suffering from acaricide failure and to overcome development of resistance in ticks. Keywords Amitraz Discriminating concentration Rhipicephalus (Boophilus) microplus Resistance
Introduction India is blessed with a bovine population of 199.10 million cattle and 105.30 million buffalo, accounting for 16.2 and 56.9 %, respectively, of world bovine populations and stands first in the world in size of bovine population (Annual Report 2011–12). Almost all
S. Kumar A. K. Sharma D. D. Ray S. Ghosh (&) Entomology Laboratory, Parasitology Division, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, UP, India e-mail: [email protected]
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of these animals are suffering from tick infestations at different times of the year. Amongst 106 valid tick species reported from India, Rhipicephalus (Boophilus) microplus is widely prevalent and is the most damaging species infesting livestock, zoo and wild animals (Ghosh et al. 2007). Economic losses to cattle producers from ticks and tick-borne diseases (TTBDS) has been estimated at US $ 13–14 milliard globally on an annual basis (de Castro 1997), to which Brazil and Australia contribute US $ 2 milliard (Grisi et al. 2002) and Aus $ 175 million (Playford et al. 2005), respectively. In India, the control cost of TTBDS in the dairy sector has been estimated at $498.7 million per annum (Minjauw and Mc Leod 2003). This tick species causes severe economic losses through anorexia, toxicosis, decreased milk yield, meat production, damaged hides and by transmission of diseasecausing agents, babesiosis and anaplasmosis (Rodrı´guez-Vivas et al. 2004; Ghosh et al. 2007). The use of different groups of acaricides viz. organophosphates (OP), synthetic pyrethroids (SP), amidines and macrocyclic lactones is the most common tick control method. However, due to continuous and indiscriminate use of insecticides, resistance has been detected in R. (B.) microplus almost every chemical that is registered for use against it (Whalon et al. 2008). In India, although cattle owners have reported inefficiency of these acaricides in field conditions, only limited data on resistance to these chemicals are currently available (Kumar et al. 2011; Vatsya and Yadav 2011; Sharma et al. 2012a; Shyma et al. 2012). Amitraz is a triazapentadiene compound, a member of the amidine chemical family introduced in 1972. It acts as a non-systemic octopaminergic agonist and has been used for the control of ticks on cattle (Garris and George 1985; Curtis 1985; Kagaruki 1996), dogs and wild animals (Pound et al. 2000; Elfassy et al. 2001; Kumar et al. 2001), parasitic mites of honey bee (Floris et al. 2001), and other ectoparasites of livestock (Curtis 1985). It was postulated that formamidine pesticides exert their toxic effect on target species by interaction with octapamine receptors of the CNS (Dudai et al. 1987), and possibly also by inhibition of monoamine oxidases (Atkinson et al. 1974; Schuntner and Thompson 1976). In India, amitraz is one of the commonly used acaricides for the control of cattle and dog ticks (Mathivathani et al. 2011) but concerted efforts have not been directed towards monitoring of resistance against the chemical acaricide. Amitraz resistance was detected in the Alfonso strain of Mexico (Soberanes et al. 2002), the Uma & Ultino strain of Australia (Kunz and Kemp 1994), and the R. (B.) microplus population of Columbia (Benavides et al. 2000), South Africa (Strydom and Peter 1999) and Brazil (Furlong 1999; Miller et al. 2002). However, except for a limited study by Singh et al. (2013), a survey has never been conducted to monitor susceptibility/resistance patterns of different Indian isolates of R. (B.) microplus against amitraz.
Materials and methods Acaricide Technical grade amitraz (99 %) was procured from AccuStandardÒ, USA, and was used for preparing a 49,500 ppm stock solution by dissolving 100 mg in 2 ml methanol. For the experimental bioassay, different aqueous concentrations of the acaricide were prepared (v/v) from the stock solutions and tested against the susceptible IVRI-I line (national registration no. NBAII-IVRI-BM-1-1998) and field isolates of R. (B.) microplus.
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Animals Weaned cross-bred (Bos taurus male 9 B. indicus female) male calves were reared in tick proof animal houses of the division of Parasitology, Indian Veterinary Research Institute and fed with calf starter, milk, concentrate mixture, wheat brans and water ad lib. Separate batches of five to six calves were used to maintain the reference susceptible IVRI-I line and field collected ticks. The calves were reared according to the guidelines of the ‘‘Committee for the Purpose of Control and Supervision on Experimentation on Animals’’, a statutory Indian body. Reference tick line The susceptible reference IVRI-I line was used as the standard to assess susceptibility/ resistance status in collected field isolates. The colony has been maintained in the Entomology Laboratory of Indian Veterinary Research Institute for the last 15 years and has not been exposed to any acaricides. The susceptibility status of the colony was monitored by periodical testing against several organo-phosphates, organo-chlorines, SP and formamidine compounds in independent bioassays. The homogeneity amongst different generations of IVRI-I line was established by uniform entomological parameters and by analyzing the sequences of 16s rDNA gene (Accession Nos. GU222462, GU323287, GU323288 (Kumar Rinesh et al. 2011). Determination of DC, LC50 and LC95 values The discriminating concentration (DC) of the test acaricide was determined performing dose-response bioassays with the IVRI-I line adopting the Adult immersion test (AIT) as mentioned by Drummond et al. (1973) and Benavides et al. (1999) with minor modifications. A serial of six concentrations of amitraz (49.5, 148.5, 247.5, 297.0, 396.0, 495.0 ppm) were prepared in distilled water from a stock solution. The engorged females of the IVRI-I line were thoroughly cleaned, dried on tissue paper and kept in Petri dishes padded with filter paper. Ticks were weighed and each replication was kept in different Petri dishes with proper markings for in vitro experiments. The pre-weighed engorged females were dipped in different concentrations of the test acaricide for 2 min and then dried in filter paper before transferring into the Petri dishes. After 24 h, ticks were transferred to the glass vials covered with cotton cloth and were kept in a precision (B.O.D.) incubator maintained at optimum conditions of 28°C and 85 ± 5 % RH. The biological parameters were monitored upto 14 days and the ticks which did not oviposit were considered as dead. The control groups were treated in similar manner in 5 % methanol as the solvent content in different concentrations of amitraz was equal to or less than 5 % level. Earlier, the toxicity of methanol against the IVRI-I line was studied and no adverse effect of dipping of ticks for 2 min in 50 % methanol was observed upto 14 days (Sharma et al. 2012b). The mortality of ticks was recorded regularly by observing the loss of motility and pedal reflex after exposing to light. The data on mortality, egg masses, RI and inhibition of oviposition (IO) were collected. The following parameters were compared: (1)
Mortality: Data were recorded upto 14 days post treatment (dpt) when normal ticks complete egg laying.
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The egg masses laid by the live ticks were recorded. RI = egg weight (EW)/ engorged female weight (IFW) treated 100 % Inhibition of oviposition ðIO % Þ ¼ RI control-RI RI control
Dose response data were analyzed by probit method (Finney 1962) using GraphPad Prism version 4.0, San Diego, CA, USA. The LC50 and LC95 values of amitraz were determined applying regression equation analysis to the probit transformed data of mortality. The differences in mean values of entomological data among the groups were analyzed by one way ANOVA. The DC was determined as 2 9 LC95 against the reference IVRI-I line (Jonsson et al. 2007). Sampling and characterization of field isolates A two stage stratified sampling method was adopted to collect samples from organized dairy farms and from villages of different districts located in the states of Uttar Pradesh, Bihar, Punjab, Rajasthan and West Bengal. Both cattle and buffalo populations were screened for tick infestations. A questionnaire was developed to collect basic information about the mode, frequency, type and dose of application of acaricides, and owners experience on efficacy of the commonly used acaricides. The following isolates : RBL, BEG, DRB, MKT, COR, BHT, SKR, JPR, ALW, NAD and N-24P were collected from both organized and unorganized farms. The areas of collection were upper Gangetic plains region situated at 25°N–26°N, 100°E–81°E (Uttar Pradesh), middle Gangetic plains region situated at 25.15°N–25.45°N, 85.4°E–86.3°E (Bihar), trans-Gangetic plains region situated at 30°N–29°N, 74°E–74°E (Punjab), western dry region situated at 23°N–25°N, 77.4°E– 78°E and central plateau and hill region situated at 25°N–27.6°N, 73°E–75.1°E (Rajasthan), the lower Gangetic plains situated at 22°N–24°N, 88°E–89°E (West Bengal). In the upper, lower and middle Gangetic plain regions the annual rainfall ranges from 900–1,500 mm with temperatures of 4–45 °C and 80–90 % humidity. In the trans-Gangetic plains, the annual rainfall ranges from 200–380 mm and the temperature from 12.8–33.9 °C with 79–85 % humidity. The western dry, central plateau and hill regions experience annual rainfall in the range of 200–650 mm with temperatures ranged from 5–45 °C and 47–74 % humidity. Engorged females of all the isolates were collected from animal sheds in separate vials, covered with cotton cloths to allow air and moisture exchange, and were transported to the laboratory. All the samples collected from a particular area were mixed and washed in water, labeled and kept at 28°C and 85 ± 5 % relative humidity. The AIT was conducted at the spot where engorged ticks were collected in a large number. The ticks were kept for laying eggs where few ticks were collected. In the laboratory, the egg masses of different engorged females collected from a particular area were pooled and pooled larvae were released on calves for feeding. After 18–20 days of feeding the engorged females were collected from the animal shed and resistance/susceptibility to amitraz was determined by AIT using statistically significant number of ticks. Resistance factors (RF) for field tick isolates were worked out by dividing LC50 of field ticks using different grades of DC i.e., 29, 49, 69 by LC50 of IVRI-I line (Castro-Janer et al. 2009). On the basis of RF, the resistance level in the field population of R. (B.) microplus was classified as susceptible (RF B 1.4), level I resistance (RF = 1.5–5.0), level II resistance (RF = 5.1–25.0), level III resistance (RF = 26–40) and level IV resistance (RF C 41) (Kumar et al. 2011).
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Results Determination of LC50 and LC95 values A direct relationship of the dose-response mortality of the IVRI-I line of R. (B.) microplus treated with different concentrations of amitraz was recorded (Fig. 1). A 100 % mortality of ticks with complete IO was recorded when treated with 495.0 ppm concentration. The Log probit model indicated a high slope value of 3.49 ± 0.46 revealing high drug response and 94 % correlation factor. However, negative slopes of egg masses (-19.97 ± 18.1) and RI (-0.22 ± 0.13) showed that both the parameters were adversely affected by the acaricide. Analyzing the log-probit equation, the LC50 was calculated as 165.0 ppm with a 95% confidence limit of 155.7–174.9 while the LC95 value was 487.7 ppm with a 95 % confidence limit of 455.8–521.8. The DC was calculated to be 975.4 ppm (Table 1). Survey areas In most of the areas surveyed, the tick activity was high from April to October and then generally declined to a minimum during winter. At a few places the farmer reported tick activity on their animals round the year. According to the veterinary records it was noted that formamidine, OP, SP and macrocyclic lactones were frequently used for tick control in both organized and unorganized farms. The farmers are using acaricides only when they see ticks on their animals. The use of acaricides in organized dairy farms was twice or thrice a month. The most common methods of acaricide application were spray, injectable route and swabbing. It was noticed that no regular acaricide treatment regime was maintained in any of the surveyed areas irrespective of the type of farms. Besides application of acaricides, natural control by birds was also seen in unorganized farms where animals were allowed for grazing in the open fields. At most of the places, the women were involved in hand picking of ticks from their animals in leisure hours.
Fig. 1 Dose-dependent mortality and inhibition of oviposition (IO) of Rhipicephalus (B.) microplus IVRI-I line treated with amitraz
Table 1 LC50 and LC95 values of amitraz obtained by adult immersion test (AIT) for the susceptible IVRI-I line of Rhipicephalus (B.) microplus Variables
Slope ± SE
R2
LC50 (ppm) (95 % CL)
LC95 (ppm) (95 % CL)
165.0 (155.7–174.9)
487.7 (455.8–521.8)
Mortality
3.49 ± 0.46
0.94
Egg mass
-19.97 ± 18.1
0.23
-0.22 ± 0.13
0.44
0.14 ± 0.04
0.70
RI % IO
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Table 2 Amitraz resistance status in field isolates of Rhipicephalus (B.) microplus Level of resistancea
Tick isolates
Slope ± SE
LC50 value (95 % CL)
Resistance factor
RBL
3.96 ± 4.32
1917.4 (1826.1–2013.2)
11.6
II
BEG
3.09 ± 1.24
825.1 (771.1–882.8)
5.0
I
DRB
6.53 ± 0.50
1815.7 (1757.8–1864.8)
11.0
II
MKT
5.31 ± 3.78
2066.8 (1983.3–2149.5)
12.5
II
COR
3.34 ± 1.084
1929.1 (1837.2–2025.5)
11.7
II
BHT
1.27 ± 0.16
401.1 (341.2–475.1)
2.4
I
SKR
3.00 ± 1.24
4501.2 (2880.6–3115.7)
27.3
JPR
2.77 ± 1.16
3847.7 (3530.0–4194.0)
23.3
III II
ALW
7.17 ± 4.04
1537.5 (1507.3–1568.2)
9.3
II
NAD
1.08 ± 0.43
99.7 (82.5–120.7)
0.6
Susc.
N-24P
1.53 ± 0.61
255.8 (224.4–291.5)
1.56
I
a
Susceptible = RF \ 1.4, level I = 1.5 \ RF \ 5.0, level II = 5.1 \ RF \ 25, level III = 26 \ RF\ 40
Amitraz resistance pattern The details of LC50 values, RF and level of resistance in different isolates are presented in Table 2. Out of eleven isolates, the maximum LC50 value of 4501.2 with confidence limit of 2880.6–3115.7 was recorded in the SKR isolate collected from large to very large organized animal farms, and frequency of use of different acaricides is very high. Although BHT, SKR, JPR and ALW isolates were collected from comparatively dry areas, the tick problem was at a very high level. The other isolates (DRB, BEG) were collected from comparatively high humid areas and the use of amitraz was practised along with that of pyrethroids at frequent intervals and the resistance factor to amitraz reached upto 11.0. The resistance to amitraz was detected in eleven isolates; resistance at level I in 3 isolates (RF = 1.56–5.0), at level II in 6 isolates (RF = 9.3–23.3) and at level III in one isolate (RF = 27.3) while the NAD isolate was found susceptible (Table 2). The regression analysis of collected tick isolates revealed that the resistant isolates of Level II and III have higher slope values. The highest steep response of amitraz was recorded in DRB isolate showing 99.4 % chance to give the regression line of 6.53 ± 0.50 and isolate came under resistance level II. Similar pattern was recorded in MKT, COR, ALW, RBL and JPR isolates. The SKR isolate with the highest resistance value exhibited 3.00 ± 1.24 slope value with R2 of 0.644 indicating that the drug response was not very high in different DCs on the isolate. The field isolates of resistance level I showed a more homogenous and gradual drug response with steep slopes and higher R2 (Table 2). In case of the reference susceptible IVRI-I line, the dose mortality response was 3.49 ± 0.46 showing a steeper slope with 0.94 R2 value.
Discussion India is the world’s largest producer of milk and dairy farming is considered an important source of income of the farmers’ community. Most dairy herds in northern India are crossbred (Bos taurus 9 B. indicus) cattle and buffaloes. The problem of TTBDS is
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particularly relevant in India because of the favorable environmental conditions for tick survival throughout most parts of the year and the maintenance of susceptible cross-bred animals to improve the production of milk and other animal products. The most widely used method for the control of ticks is the direct application of acaricides to host animals and thus the consumption of insecticides has been increased manifold during the last decades. Amitraz, a formamidine acaricide, plays an important role in the control of the cattle tick, R. (B.) microplus, and other tick species that infest cattle and pet animals. It has minimal toxicity to cattle or humans and no meat withholding period, making it suitable for use in cattle shortly before slaughter (Jonsson and Hope 2007). Although amitraz resistance in R. (B.) microplus was previously reported in several countries, the actual field situation in India has not been worked out due to non-availability of a suitable bioassay technique and the DC required for the detection of resistance. The unavailability of engorged female ticks in sufficient number required to do AIT for the diagnosis of drug resistance is the technical limitation of this bioassay (Jonsson et al. 2007). In response to the lack of a linear response to amitraz in the standard LPT, Thullner et al. (2000) described a dispersal test using a paper grass stem model. This model provided linear responses to amitraz but has not been widely adopted. The larval packet test was further modified by the introduction of formulated commercial acaricide in place of technical product and using a nylon material in place of filter paper (Miller et al. 2002) and (Ducornez et al. 2005). In the Entomology laboratory of IVRI, AIT has been effectively standardized using technical grade insecticides and DCs for deltamethrin, cypermethrin, malathion, diazinon, fipronil, coumaphos and fenvelerate were worked out with repeatability. The technical grade insecticides were selected over commercial formulation because commercial products are prepared with many proprietary ingredients and it is difficult to assess the responses due to active ingredients (Shaw 1966).The response obtained using technical grade insecticides was linear and was effectively used for characterization of ticks collected from six agroclimatic zones of India (Sharma et al. 2012b). Using the same methodology, in the present study, the DC of amitraz was determined effectively as 975.4 ppm using reference IVRI-I lines of R. (B.) microplus. Presently, the working group on parasite resistance recommends AIT (Drummond et al.1973) for the detection of resistance since it is fast, simple and a relatively cheap method to be adopted in the field (FAO 2004). The AIT method has been successfully adopted in Australia (Jonsson et al. 2007), Brazil (Brito et al. 2011) and in India (Kumar et al. 2011; Sharma et al. 2012b;.Kumar Rinesh et al. 2013; Singh et al. 2013) for the detection of resistance to SP, OP, moxidectin and amitraz. The current study reports the resistance status of R. (B.) microplus against amitraz and the mechanism involved needs to be established. The mode of action of amitraz is believed to be interference with nervous system function of the targeted species by binding to the octopamine receptors (Evans and Gee 1980). Several types of octopamine receptors including a putative octopamine- like, G-protein-coupled receptor were identified in insects (Blenau and Baumann 2001), and in R. (B.) microplus (Baxter and Barker 1999), respectively. However, past reports of involvement of metabolic detoxification and mutation of the octopamine receptors were speculated to be the main mechanism of resistance to amitraz (Li et al. 2004). The use of amitraz became more frequent when pyrethroid resistance problems started to hinder the tick control efforts in Mexico. The first case of amitraz resistance was detected in the San Alfonso strain of R. (B.) microplus collected from a ranch in the state of Tabasco (Soberances et al. 2002). Reports of resistance against amitraz are available against R. (B.) microplus ticks from various parts of the world (Jonsson et al. 2000; Li et al.
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2004; Veiga et al. 2012) and the factors associated with increased probability of R. (B.) microplus resistance in Australia were identified as region, frequency, doses and application method (Jonsson et al. 2000). In India, the presence of resistance to different families of chemical acaricides has already been reported by many workers (Sangwan et al. 1993; Kumar et al. 2011; Vatsya and Yadav 2011; Sharma et al.2012b; Shyma et al. 2012; Kumar Rinesh et al. 2013). Recently, Singh et al. (2013) reported gradual development of amitraz resistance in R. (B.) microplus collected from Gujarat state of India. The present study supported the views of Singh et al. (2013) with additional information on development of multi-acaricide resistant tick populations in the areas of collection since all these isolates were found resistant to a number of OP and SP compounds, possible reasons for acaricide failure in many parts of the country. Acknowledgments The authors are grateful to Indian Council of Agricultural Research, New Delhi for funding through World Bank funded National Agricultural Innovation Project No. NAIP/Comp-4/C2066/ 2008-09. Authors are also grateful to the Veterinary officers posted at different tick collection spots for their support.
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