Veterinary Parasitology

Veterinary Parasitology 181 (2011) 274 281 Contents lists available at ScienceDirect Veterinary Parasitology jou rn al h om epa ge: www.elsevier.com/locate/vetpar Diazinon resistant status in Rhipicephalus (Boophilus) microplus collected from different agro-climatic regions of India Sachin Kumar a, Souvik Paul a, Anil Kumar Sharma a, Rinesh Kumar a, Shashi Shankar Tewari c, Pallab Chaudhuri b, D.D. Ray a, Ajay Kumar Singh Rawat c, Srikant Ghosh a, a Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, UP, India b Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Bareilly-243122, UP, India c Division of Pharmacognosy & Ethnopharmacology, National Botanical Research Institute, Rana Pratap Marg, P.B. 436, Lucknow-226001, UP, India a r t i c l e i n f o Article history: Received 1 February 2011 Received in revised form 7 April 2011 Accepted 14 April 2011 Keywords: Rhipicephalus (Boophilus) microplus Diazinon Resistance Agro-climatic regions India a b s t r a c t The resistance status of Rhipicephalus (Boophilus) microplus to Diazinon was evaluated in 20 locations situated at various agro-climatic regions of India. Adult immersion test (AIT) was optimized using laboratory reared acaricide susceptible IVRI-I strain of R. (B.) microplus and minimum effective concentration of Diazinon was determined as 635.2 ppm. The discriminating dose (DD) was worked out as 1270.4 ppm and was tested on female ticks collected from organized and unorganized farms located at different agro-climatic regions of India. On the basis of the data generated on three variables viz., mortality, egg masses and reproductive index, the resistance level was categorized as I, II, III and IV. The average resistance factor (RF) of 6.1 (level II) was recorded in the ticks collected from the northern sub-temperate trans-gangetic plains while high average RF values of 26.65 (level III) was recorded in the ticks collected from tropical middle-gangetic plains. The tropical middle gangetic plain has a very high density of animal populations where farmers use Diazinon for tick control, for agricultural practices and for mosquito control. Due to the continuous use of OP compounds the environmental load of Diazinon has become high in the area. This is the first experimental data generated on Diazinon resistant status in ticks of India. 2011 Elsevier B.V. All rights reserved. 1. Introduction Amongst the 106 valid tick species reported from India, Rhipicephalus (Boophilus) microplus is widely prevalent and considered as the most economically important tick infesting livestock (Ghosh et al., 2007). Besides avid blood suckers, this tick species act as the vector of bovine babesiosis and anaplasmosis and also cause 20 30% reduction in the cost of leather due to tick bite marks (Biswas, 2003). Control of ticks is focused on large scale repeated use of acaricides viz., organophosphates (OP), synthetic pyrethroids Corresponding author. E-mail address: sghoshp@yahoo.co.in (S. Ghosh). (SP), amidines and macrocyclic lactones (Khan, 1996; Kemp et al., 1999; Bianchi et al., 2003; Rodriguez-Vivas et al., 2007) with limited success. Repeated application of these chemicals leads to the development of resistance in the ticks which is considered as the main hindrance for successful pest and vector control program in livestock globally (Shidrawi, 1990; FAO, 2004; Graf et al., 2004). According to Whalon et al. (2008), R. (B.) microplus developed resistance to almost every chemicals registered for use against it and ranked sixth amongst the resistant arthropods. In India, about 60% of livestock is reared by small and marginal farmers and use of OP compounds like diazinon and malathion is very common for the control of agricultural pests including livestock and poultry (Sharma, 0304-4017/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.04.030

S. Kumar et al. / Veterinary Parasitology 181 (2011) 274 281 275 2004; Ghosh et al., 2006). Besides their applications against agriculturally important pests, OP compounds are also used for mass eradication of mosquito larvae in the breeding places (ICMR Bulletin, 2002). Although farmers have complaints about lack of efficacy of different chemical acaricides against ticks, validation of acaricide resistance with suitable in vitro bioassays for generating base line informations has not been done. In the present study, the status of acaricide resistance against the organophosphate compound Diazinon in targeted populations of R. (B.) microplus infesting cattle in different agro-climatic regions of India was evaluated by standardizing an adult immersion test (AIT). 2. Materials and methods 2.1. Acaricide Technical grade diazinon (100% pure) was procured from AccuStandard Inc. U.S.A., and was used for preparation of 50,000 ppm stock solution in methanol. For the experimental bioassay, different concentrations of the acaricide were prepared in distilled water from the stock solution and tested against R. (B.) microplus. 2.2. Animals Weaned crossbred (Bos taurus male B. indicus female) male calves were reared in tick proof animal house 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 acaricide susceptible IVRI-I strain of R. (B.) microplus and field collected ticks. The calves were maintained as per the guidelines of Committee for the purpose of control and supervision on experimentation on animals (CPCSEA), a statutory Indian body. 2.3. Ticks The colony of acaricides susceptible reference IVRI- I strain of R. (B.) microplus was used as the standard to assess resistance. The colony is maintained in the Entomology Laboratory of Indian Veterinary Research Institute for the last fifteen years and had not been exposed to any acaricides. The susceptibility status of the colony was established by periodic testing against several organophosphates, organochlorines, synthetic pyrethroids and formamidine compounds in independent bioassays. The genetic homogeneity amongst different generations of IVRI I strain has been established by uniform entomological data and by analyzing the sequences of 16s rrna gene of the tick species (GenBank accession nos. GU222462, GU323287, GU323288). 2.4. Adult immersion test (AIT) Adult immersion test was conducted according to Drummond et al. (1973) and Benavides et al. (1999) with some modifications. 2.4.1. Optimization of immersion time To optimize the immersion time, a control experiment was conducted in which pre-weighed engorged females of susceptible IVRI-I strain (n = 30 in each duration of time, three replication each containing ten ticks) were immersed in 350 ppm concentration of cypermethrin, diazinon and malathion for different time periods viz., 30 min, 20 min, 10 min, 5 min and 2 min. Control ticks were treated with distilled water and maintained simultaneously. After immersion, the ticks were soaked in filter paper and then transferred into the Petri dishes padded with Whatman filter paper. The mortality pattern was noted. The minimum immersion time at which 100% ticks stopped egg laying was determined and used for standardization of bioassay for determination of DD of diazinon. 2.4.2. Bioassay For the generation of base line concentration of diazinon different concentrations viz., 100, 150, 200, 250, 300, 350, 400, 450 500, 600 and 650 ppm were prepared in distilled water from the 50,000 ppm stock solution in methanol. The pre weighed engorged females of IVRI-I strain of R. (B.) microplus were immersed in different concentrations of diazinon for an optimized time, soaked in filter paper before transferring into the Petri dishes. After 24 h, ticks were transferred to the glass tubes covered with muslin cloth and were kept in desiccators placed in BOD incubator maintained at 28 ± 1 C and 85 ± 5% RH. The mortality of ticks was recorded by observing loss of motility and pedal reflex after exposing to light. The control ticks were treated in similar manner in 10% methanol as the methanol content in different concentrations of acaricide was equal to or less than 10% level. Each concentration was replicated 10 times and five adults were used per replication (n = 10 5). The safety level of methanol against susceptible IVRI I strain of R. (B.) microplus has previously been evaluated up to the level of 50% (Sharma et al., 2011). The following parameters were compared: (a) Mortality: data were recorded up to 14 dpt (days post treatment) when normal ticks completed their egg laying. (b) The egg masses laid by the ticks were recorded. (c) Reproductive index (RI) = egg weight (EW)/engorged female weight (IFW). (d) Percentage inhibition of oviposition (IO%) = ((RI control RI treated)/ri control) 100. 2.5. Collection of ticks from different agro-climatic regions Two stage stratified sampling method was adopted to collect live engorged females of R. (B.) microplus ticks from animals and from the sheds. Both organized and unorganized farms were surveyed to collect the samples. The areas of collection were selected from six different agro-climatic regions of India (India has 15 agro-climatic regions) where intensive agricultural and animal husbandry activities including regular application of acaricides on cattle is practiced. The areas of collection were trans-gangetic plain region in the state of Punjab (Ludhiana, Bhatinda,

276 S. Kumar et al. / Veterinary Parasitology 181 (2011) 274 281 Fig. 1. Collection of tick samples from different agro-climatic regions (shaded) of India. Patiala, Muktsar) (region 6), western dry region in the state of Rajasthan (Chittorgarh, Banswara) (region 14), upper gangetic plains in the state of Uttar Pradesh (Bareilly, Raebareilly) (region 5), middle gangetic plains in the state of Bihar (Patna, Muzaffarpur, Begusarai, Danapur, Darbhanga, Sultanpur, Vaishali) (region 4), lower gangetic plains in the state of West Bengal (Nadia, 24-Parganas (south), 24- Parganas (north) (region 3), and eastern himalayan region in the state of Tripura (west Tripura) (region 2) (Fig. 1). The states of Punjab, Uttar Pradesh, Tripura and Bihar are humid sub-tropical areas, the state of West Bengal is a tropical wet area while Rajasthan is a arid dry region of the country. A questionnaire was formulated to collect the data on frequency and type of acaricide treatment adopted by the respondents/cattle owners. The ticks were collected in separate vials closed with muslin cloth to allow air and moisture exchange and kept at 28 ± 1 C and 85 ± 5% relative humidity. 2.6. Tick control practices 2.6.1. Unorganized farms In all the nineteen locations, about 85% of the animals were cross bred cattle dedicated to milk production and were heavily to moderately infested with ticks. The level of tick infestation was comparatively higher in cross-bred animals in comparison to Zebu breed of animals which are mainly maintained for draft purposes. Regarding the various tick control practices, hand picking was reported as one of the most common methods adopted by small and marginal farmers. However, natural control by birds and crows was also seen in unorganized farms as animals were let loose for grazing in the open fields. The frequency of acaricides application was comparatively less than organized farms. The use of OP compounds was more frequent in agricultural fields and the same insecticides were generally used for tick control. The use of acaricides was comparable

S. Kumar et al. / Veterinary Parasitology 181 (2011) 274 281 277 in all the areas surveyed and expressed as the mean percentage of farmers using tick control as 24.5 42.0% (routine treatment), 36.8 65.4% (occasional treatment), and 3.7 18.0% (no treatment). The farmers using acaricides only when they see ticks on their animals and largely contributed to the No treatment group. Swabbing was the most common method of application of acaricides for tick control. 2.6.2. Organized farms Buffaloes and cross bred cattle were reared for milk production. The frequency of acaricides application for the control of the tick populations in these farms was very high and the mode of application was spray, injectable route and pour-on. Amongst the respondents 34% used pyrethroids, 22% ivermectin, 28% organophosphates and 16% flumethrin during the last five years. 2.7. Screening of field tick for the detection of resistance After determining the LC 95 value of diazinon against susceptible IVRI-I strain, different discriminating doses (DD) viz., 2x, 4x, 6x, 8x, etc. were prepared in distilled water from the stock solution of diazinon where x is the calculated LC 95. The larvae emanated from female ticks collected from fields were fed on disease free cross bred calves maintained in the large animal house of the division of Parasitology of the institute to obtain sufficient number of adult ticks in the next generation. The engorged adult ticks were used in AIT as per the method mentioned above. On an average 200 250 ticks were used in different discriminating doses for characterization of resistance. 2.8. Statistical analysis Dose response data were analyzed by probit method (Finney, 1962) using GraphPad Prism 4 software. The LC 50 and LC 95 values of diazinon were determined by applying regression equation analysis to the probit transformed data of mortality. For characterization of field ticks, discriminating dose (DD) was determined as 2 LC 95 of IVRI-I strain (Jonsson et al., 2007). Resistance factors (RF) for field isolates were worked out by the quiescent between LC 50 of field isolates and LC 50 of IVRI-I strain of R. (B.) microplus (Castro-Janer et al., 2009). On the basis of RF, the resistance status in the field population of R. (B.) microplus was classified as susceptible (RF < 1.4), level I resistance (1.5 < RF < 10.0), level II resistance (10.1 < RF < 25.0), level III resistance (26 < RF < 40) and level IV resistance (RF > 41). The differences in mean values of entomological data amongst the groups were analyzed by one way ANOVA (Snedecor and Cochran, 1968). 3. Results 3.1. Optimization of immersion time Table 1 shows the effect of different immersion times on mortality of ticks treated with three different acaricides. Comparing the mortality data, it was observed that different immersion times have no differential effect on tick biology. Accordingly, the minimum immersion time was determined as 2 min for determination of DD of diazinon and to characterize the filed collected ticks. 3.2. Minimum effective concentration of diazinon The data on minimum effective concentration of diazinon against IVRI-I strain of R. (B.) microplus is shown in Table 2. The mortality of ticks was reduced with decreasing concentrations of diazinon and 100% mortality of ticks was recorded at 650 ppm. Reproductive index (RI) of survived ticks was found to be significantly inhibited in dose dependent manner. The highest reduction in RI was noticed in 650 ppm followed by 600 ppm at which a RI of 0.156 ± 0.24 was noted and the data were found statistically significant (p < 0.001) in comparison to control (0.415 ± 0.02). A 100 ppm concentration did not show any significant alteration in RI and in IO%. The value of LC 50 was calculated as 372.0 ppm with a 95% confidence limit of 341.28 405.28 while the LC 95 value was 635.2 ppm with a 95% confidence limit of 582.75 692.37. The DD for assessment of resistance status of field ticks was calculated 1270.4 ppm. 3.3. Resistance status The LC 50 value of diazinon and the level of resistance in the field isolates are shown in Table 3. The two field isolates (Bareilly and Patna) were identified as field susceptible. The resistance level in the three isolates (Vaishali, Sultanpur and Banswara) was determined at level IV, while the resistance level in Danapur and Muzaffarpur isolates was at III. The RF value of other isolates collected from the different agro-climatic zones was grouped either in level I or II. The average RF values were lowest in the trans-gangetic regions (6.1) and the highest (26.65) in the middle-gangetic regions (Table 3). The middle gangetic region possesses very high population of animals and the farmers are using organophosphate compounds for the control of agricultural pests including ticks and also for the control of mosquito borne human diseases. The egg masses produced by IVRI-I strain and ticks showing resistance at levels I IV are shown in Fig. 2. The slope of the egg masses of the ticks showing resistance level IV was lowest in comparison to other ticks. It was observed that between resistant levels I and III there was no significant effect on production of egg masses in dose dependent manner. The R 2 values of the field resistant ticks for egg masses and reproductive index were recorded in the range of 0.61 1.0 and 0.57 0.80, respectively. The R 2 values of the egg masses and RI of the susceptible ticks were of 0.64 and 0.75, respectively (Table 4). 4. Discussion The standard bioassay recommended by FAO for testing resistance to acaricides in R. (B.) microplus is the larval packet test (LPT) originally described by Stone and Haydock (1962). The LPT takes 5 6 weeks to complete, is a laborious test and requires significant laboratory resources to conduct the test routinely. While AIT can be conducted in ease and data can be generated within two weeks time. Standardization of AIT for field depends on the identification

278 S. Kumar et al. / Veterinary Parasitology 181 (2011) 274 281 Table 1 Effect of different immersion times on mortality of IVRI-I strain of R. (B.) microplus. Acaricides Immersion time (min) Tick wt. (mg) % Mortality 10 dpt Final Diazinon (350 ppm) Cypermethrin (350 ppm) Malathion (350 ppm) Control 2 119.6 ± 7.0 26.7 ± 4.2 41.3 ± 6.1 5 114.3 ± 5.7 33.3 ± 4.2 40.0 ± 5.2 10 125.6 ± 3.6 26.7 ± 4.2 40.0 ± 5.2 20 117.0 ± 5.0 30.0 ± 4.5 36.7 ± 8.0 30 114.1 ± 4.5 30.0 ± 6.8 43.3 ± 8.0 2 118.6 ± 2.9 73.3 ± 4.2 100.0 ± 0.0 5 128.8 ± 3.8 73.3 ± 4.2 100.0 ± 0.0 10 119.8 ± 2.8 80.0 ± 7.3 96.7 ± 3.3 20 128.4 ± 2.4 73.3 ± 8.4 96.7 ± 3.3 30 126.4 ± 5.4 80.0 ± 7.3 96.7 ± 3.3 2 135.2 ± 5.2 10.0 ± 6.8 16.7 ± 6.1 5 123.5 ± 3.5 10.0 ± 4.5 13.3 ± 4.2 10 122.8 ± 3.7 6.7 ± 4.2 16.7 ± 8.0 20 127.8 ± 2.2 10.0 ± 6.8 13.3 ± 6.7 30 134.1 ± 4.6 6.7 ± 4.2 16.7 ± 6.1 2 119.6 ± 7.0 0 ± 0 5.0 ± 0 5 117.0 ± 5.0 0 ± 0 0 ± 0 10 126.4 ± 5.4 0 ± 0 10.0 ± 0 20 123.5 ± 3.5 0 ± 0 5.0 ± 0 30 134.1 ± 4.6 0 ± 0 0 ± 0 Table 2 Dose dependent response of diazinon against IVRI-I strain d of R. (B.) microplus. Conc. (ppm) Tick weight (mg) (mean ± SE) Final % mortality (mean ± SE) Egg mass (mg) (mean ± SE) RI (mean ± SE) % IO 100 101.9 ± 1.4 4.0 ± 2.7 39.2 ± 1.7 b 0.387 ± 0.015 6.7 150 103.0 ± 1.0 16.0 ± 5.8 28.9 ± 2.1 c 0.279 ± 0.014 b 32.8 200 102.2 ± 0.5 24.0 ± 8.3 23.9 ± 2.7 c 0.218 ± 0.028 c 47.5 250 102.5 ± 0.5 34.0 ± 5.2 b 20.8 ± 1.8 c 0.209 ± 0.024 c 49.8 300 1103.6 ± 1.0 38.0 ± 5.5 c 20.9 ± 1.9 c 0.196 ± 0.011 c 52.8 350 103.3 ± 1.0 40.0 ± 5.2 c 17.8 ± 1.5 c 0.189 ± 0.031 c 54.4 400 104.4 ± 0.6 46.0 ± 5.2 c 21.6 ± 1.6 c 0.200 ± 0.008 c 51.8 500 115.3 ± 4.8 74.0 ± 5.2 c 24.7 ± 1.3 c 0.189 ± 0.002 c 54.4 600 112.0 ± 5.0 92.0 ± 3.3 c 18.5 ± 2.5 c 0.156 ± 0.024 c 62.4 650 112.0 ± 5.0 100 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c 100.0 Control 110.5 ± 9.8 4.0 ± 2.7 49.3 ± 3.4 0.415 ± 0.02 b Significant at p < 0.01. c Significant at p < 0.001. d Reference susceptible srain. Table 3 Slope, LC 50, 95% CL and RF values of diazinon against R. (B.) microplus collected from different agro-climatic regions of India. Agroclimatic region Tick isolates Slope ± SE LC 50 values (95% CL) RF a Level of resistance Upper gangetic plains IVRI-I strain 7.06 ± 0.54 372.0 (341.28 405.28) 1.0 Susceptible Raebareilly 1.61 ± 0.52 7033.0 (6336.1 7806.7) 18.9 II Bareilly 2.94 ± 1.18 398.7 (379.7 418.6) 1.07 Susceptible Middle gangetic plains Danapur 0.89 ± 0.36 14059.1 (11619.1 17011.5) 37.8 III Muzaffarpur 1.12 ± 0.89 11486.8 (10538.3 21824.9) 30.9 III Begusarai 2.46 ± 1.19 1332.8 (1245.6 1426.1) 3.6 I Vaishali 1.23 ± 0.34 15313.2 (15161.6 15466.3) 41.2 IV Derbhanga 1.93 ± 1.54 5381.9 (5328.6 5435.7) 14.5 II Sultanpur 1.13 ± 0.45 21522.3 (18553.7 24965.8) 57.5 IV Patna 3.07 ± 0.33 382.5 (364.3 401.6) 1.03 Susceptible Trans gangetic plains Patiala 1.40 ± 0.19 1942.5 (1719.0 2195.0) 5.2 II Bathinda 3.51 ± 0.07 1943.9 (1851.3 2041.1) 5.2 II Ludhiana 2.34 ± 0.36 1543.8 (1442.8 1651.9) 4.2 I Muktsar 3.25 ± 0.94 3655.6 (3384.8 3948.0) 9.8 II Western dry region Chittorgarh 1.18 ± 0.95 3655.6 (3384.8 3948.0) 9.8 II Banswara 1.38 ± 0.47 24545.9 (21722.0 27736.9) 66.0 IV Lower gangetic plains Nadia 4.55 ± 0.24 2417.6 (2347.2 2490.1) 6.5 II 24-Parganas (north) 3.42 ±.0.84 4360.1 (4074.8 4665.3) 11.7 II 24-Parganas (south) 2.0 ± 0.31 1845.44 (1693.0 2011.5) 5.0 II Eastern Himalayan region West Tripura 2.22 ± 0.82 4170.8 (3861.8 4504.5) 11.2 II a RF, resistance factor.

S. Kumar et al. / Veterinary Parasitology 181 (2011) 274 281 279 Table 4 Comparison of egg masses and reproductive index of susceptible and resistant R. (B.) microplus against diazinon at different levels. Tick isolates Level of resistance RF value Variables Slope SE slope R 2 IVRI-I strain Reference susceptible 1.0 Egg mass 29.65 7.88 0.64 RI 0.32 0.06 0.75 Begusarai I 3.6 Egg mass 22.67 0.65 1.0 RI 0.79 0.04 0.80 Raebareilly II 18.9 Egg mass 20.56 11.55 0.61 RI 0.16 0.10 0.57 Danapur III 37.8 Egg mass 3.44 0.70 0.96 RI 0.04 0.02 0.75 Banswara IV 66.0 Egg mass 36.76 13.02 0.66 RI 0.11 0.03 0.74 Egg masses (mg) of DD, above which no susceptible adults are able to lay eggs and below which few or no resistant ticks are killed. In the present study, using a 14 day oviposition protocol, DD for diazinon has been worked out using susceptible IVRI-I strain and was found consistent. Although, Sabatini et al. (2001) reported a seven day oviposition protocol for AIT to identify DD for macrocyclic lactones, we got consistent results when egg masses were measured on 14 dpt. In AIT, technical grade diazinon was 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). For the preparation of stock solution technical grade diazinon was dissolved in 100% methanol and the working concentrations were prepared using water. Use of a suitable organic solvent is one of the important steps in the process of preparation of the various dilutions of the acaricide. Inclusion of organic solvents also facilitates adsorption of compound over the surface area of target biological materials and possibly enhances penetration of active ingredients of the acaricide across the exoskeleton. However, organic solvent may cause toxic effects on their own and can interact with pesticides to alter their toxicity when used at 100% concentration (Stratton and Corke, 1981; Goncalves et al., 2007). In a separate control study, the safety level of methanol against susceptible IVRI-I strain of R. (B.) microplus was determined up to the level of 50% 125 100 75 50 25 Susceptible Begusarai (Level I) Raebareilly (Level II) Danapur (Level III) Banswara (Level IV) 0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 log conc. (ppm) Fig. 2. Comparative pattern of egg masses laid by ticks having different levels of resistance and IVRI-I strain of R. (B.) microplus. (Sharma et al., 2011). In the present study, the methanol concentration did not exceed 10% level in any of the dilutions of diazinon. In AIT, mortality was used as the main criterion for calculation of LC 50 and LC 95 values of the IVRI-I strain and field isolates because this was the earliest parameter to measure and the chances of data variation was low as the number of ticks used were large (n = 1100 for IVRI-I strain). Absence of repetition in AIT values has been attributed to great data variation in low sample sizes likely to be obtained directly from the field (Jonsson et al., 2007). To neutralize the variation in the present study, field ticks were colonized to enhance the sample size and on an average 250 300 adult ticks of each isolate were tested. The immersion time is a critical factor which influenced the out come of AIT (Mendes et al., 2000; Oliveira et al., 2000; Castro-Janer et al., 2009). Although FAO (2004) prescribed immersion time of 30 min for AIT, Castro-Janer et al. (2009) tested different DD of fipronil against R. (B.) microplus by 1 min immersion time. Before conducting the bioassay for diazinon, a control study was conducted and minimum immersion time of 2 min was optimized as the mortality pattern of treated ticks was found to have no relationship with the changes of immersion times from 30 min to 2 min (Table 1). Previously, Sabatini et al. (2001) conducted a control study on effect of immersion time on egg laying of R. (B.) microplus and no significant changes were noted when ticks were treated with either 15 s to 30 min. The age and condition of ticks prior to AIT are likely to play an important role to variability of results (Jonsson et al., 2007). These factors were standardized in repeated laboratory experiments and consistent results were obtained. The slope of mortality was steeper in IVRI-I strain in comparison to resistant ticks which inferred that for each unit increase in concentration of the acaricide there is a greater mortality amongst the ticks of IVRI-I strain. Although AIT has been recommended by FAO (1997) for detection of resistance, contradictory results have been reported regarding the usefulness of AIT in standardization of DD for different acaricides. For example, Jonsson et al. (2007) evaluated AIT as bioassay technique to discriminate amitraz and cypermethrin resistant strains, but did not obtain reproducibility and the slope for dose response curve was very low. They were of the opinion that the results of the experiment could be improved by increasing the number of replications and number of ticks per replication. In the present experiments, DD was worked

280 S. Kumar et al. / Veterinary Parasitology 181 (2011) 274 281 out using more number of ticks and multiple repeats of each bioassay. Similarly, Castro-Janer et al. (2009) used four variables in AIT e.g., mortality, egg weight, index of fertility and index of fecundity to determine the DD for fipronil and found the test was efficient to discriminate a fipronil susceptible from resistant strain. In the present study, presence of engorged females laying eggs at DD is a positive resistant diagnostic and using this parameter it was possible to obtain repeatable data with less work and without determining percentage of hatching which is a very laborious method and cannot be conducted in the field situation. Amongst the field isolates, lowest mortality slope value was detected in Danapur isolate although it was coming under level III resistance. A consistently low mortality slope value of 1.23 1.93 was determined in ticks with level IV resistance. The data showed a very high level of heterogeneity in the field isolates in relation to diazinon resistance irrespective of different agro-climatic zones of the country. Mendes et al. (2007) categorized ticks of Sao Paulo, Brazil on the basis of RF as level I (1.5 < RF < 4.4), level II (4.5 < RF < 50), level III (RF > 50) but the range of level III was very high and not reflecting actual resistance condition in the field situation. Accordingly a separate classification suitable to Indian situation is proposed in this study. The dose response curves for egg masses, reproductive index and IO% of IVRI-I strain R. (B.) microplus were also validated by AIT. The slope of egg masses was negative because with the increasing concentrations of acaricide the ticks died. The field isolates with highest resistant factor (Banswara) showed the lowest slope of the egg masses and RI (Table 3) and thus provide ample information on development of alarming level of resistance in ticks against OP compounds in this area of Rajasthan. The DD of diazinon for field ticks was calculated as 1270.4 ppm which was in accordance with WAARC recommended DD of diazinon (0.1 0.2% or 1000 2000 ppm). The DD of the OP compound chlorpyriphos was calculated as 622 ppm on Brazilian Mozo srain of R. (B.) microplus (Mendes et al., 2007). But the FAO recommended DD for diazinon in AIT against R. (B.) microplus was 5 g/l (5000 ppm) which was very high. This may be possibly due to the fact that this DD was calculated on a strain from Mexico where resistance to OP is widespread and therefore, the thresholds of the Mexican strains were high. Moreover, it was suggested that these DD values require modification and revision in future (FAO, 2004). The results of the present study revealed the presence of a widespread resistance to diazinon in ticks collected from different locations. The confidence intervals of the field isolates were very wide suggesting the heterogeneity in the field population. Although the number of places in each zone was not same, the average RF to diazinon was highest in the middle-gangetic plain where use of organophosphate, both as agricultural pesticide and acaricide is intensively practiced. The RF of ticks collected from Sultanpur situated in this zone was 57.5 which is at very high level and precautionary guidelines required to be issued at implementation level to restrict the use of OP compounds in the region. The average RF to diazinon was lowest in the state of Punjab where majority of farmers stopped use of OP compounds more than ten years ago and introduced deltamethrin for tick control. As reported by Drummond et al. (1973), Ong are et al. (1985) and Castro-Janer et al. (2009), the present study indicated that AIT with different discriminating dosages provide an effective estimate for diazinon resistance in R. (B.) microplus ticks. This is the first experimental data generated on diazinon resistance status in ticks collected from different agro-climatic regions of India and will help in formulating suitable control measures against ticks and tick-borne diseases of animals. Acknowledgement 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. References Benavides, O.E., Romero, N.A., Rodriguez, J.L., Silva, Z.J., 1999. Evidencia preliminar de la aparicion de resistenciaa lactonas macrociclicas en cepas de garrapata Boophilus microplus en Colombia. In: Memorias IV Seminarios Internacional de Parasitologia, Puerto Vallarta, Jalisco., Mexico 20 22 (1999 October), 266 264. Bianchi, M.W., Barre, N., Messad, S., 2003. 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