Ticks and Tick-borne Diseases 5 (2014) 90–94
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Original article
A comparative study on cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum from Punjab (India) Nirbhay K. Singh a,∗ , Jyoti a , Manjurul Haque a , Harkirat Singh a , Shitanshu S. Rath a , Srikant Ghosh b a Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141004, India b Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, UP, India
a r t i c l e
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Article history: Received 11 March 2013 Received in revised form 26 August 2013 Accepted 26 August 2013 Available online 16 November 2013 Keywords: Cypermethrin Hyalomma anatolicum Punjab Resistance Rhipicephalus (Boophilus) microplus
a b s t r a c t A study to evaluate cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Muktsar and Mansa districts of Punjab state, India, was conducted by using adult immersion test (AIT). The regression graphs of probit mortality of ticks plotted against log values of concentrations of cypermethrin was utilized for the determination of slope of mortality, lethal concentration for 50% (LC50 ), and the resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index, and percentage inhibition of oviposition), the resistance levels were categorized. Resistance to cypermethrin was categorized as level II and I in R. (B.) microplus collected from Muktsar and Mansa districts, respectively, whereas, H. anatolicum from both locations showed a susceptible status. The RF values of Muktsar and Mansa field samples of engorged R. (B.) microplus (5.48 and 2.18, respectively) were much higher as those of engorged H. anatolicum (1.12 and 0.82, respectively) indicating a lower level and slower rate of development of cypermethrin resistance in multi-host ticks. The data generated in the current study might be of immense help in formulating suitable control measures against ticks and tick-borne diseases of animals. © 2013 Elsevier GmbH. All rights reserved.
Introduction Ticks and tick-borne diseases are a major problem to livestock health worldwide. Their significance depends on the region, the species involved, the host population(s) involved, and socioeconomic and technological advances in control measures (Solis, 1991). Losses attributable to ticks are caused either directly by tick bites (Biswas, 2003; Jongejan and Uilenberg, 2004), blood loss, damage to hides and udders, decreased milk yield (Sutherst, 1983; Sajid et al., 2007), or indirectly through mortality or morbidity caused by the pathogens transmitted by or associated with the ticks. The global economic loss due to tick infestation has been estimated as US$14,000–18,000 million annually, and the cost of management of ticks and tick-borne diseases in livestock of India is as high as US$498.7 million per annum (Minjauw and Mc Leod, 2003). Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum are widely prevalent and considered as economically important ixodid ticks infesting dairy animals in India (Ghosh et al., 2007), particularly in Punjab state (Haque et al., 2011; Singh and Rath, 2013).
∗ Corresponding author. Tel.: +91 9417336332; fax: +91 1612400822. E-mail address: [email protected] (N.K. Singh). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.08.002
Presently, the use of chemical acaricides is the most widely employed tick control strategy in India. These chemicals are easily available and are applied on infested animals at frequent intervals leading to their indiscriminate use which has probably contributed to the development of acaricide resistance in ticks (Sharma et al., 2012). Synthetic pyrethroids (SPs), particularly cypermethrin, is the predominantly used commercially available acaricide employed for tick control in Punjab state, India. Beside its application against agriculturally important pests, it is also used extensively for the control of mosquitoes (Ansari and Razdan, 2003; Sharma et al., 2004; Tiwari et al., 2010). Although cattle owners have reported treatment inefficiency in field conditions, only limited data on tick resistance to cypermethrin are currently available from India (Kumar et al., 2006; Sharma et al., 2012; Shyma et al., 2012) and particularly from Punjab state (Singh et al., 2010, 2013). Further, most of the published data available are regarding the resistance status of R. (B.) microplus, whereas, reports on the resistance status of H. anatolicum are scarce, and no data are available on a comparative basis of the resistance status of single-host versus multi-host ticks. Having in mind the variation in the development of acaricide resistance in different tick types, it becomes essential to generate data for these different tick species for formulation and implementation of effective tick control strategies particularly for countries like India where mixed tick
N.K. Singh et al. / Ticks and Tick-borne Diseases 5 (2014) 90–94
infestation is a common feature. The current study was undertaken to generate data on the cypermethrin resistance status of R. (B.) microplus and H. anatolicum collected from Punjab state, India. Materials and methods Study area Live engorged adult female R. (B.) microplus and H. anatolicum ticks were collected from cross-bred dairy animals with mixed infestation of both tick species as well as their sheds, from Muktsar and Mansa districts located at the trans-gangetic plain in the state of Punjab (29.30◦ N to 32.32◦ N and 73.55◦ E to 76.50◦ E) with humid subtropical climatic conditions. Besides, uniform tick infestation patterns, easy accessibility promoted the selection of these regions. Both organized and unorganized farms were selected to collect the samples. A questionnaire was formulated to collect data on frequency, type, and mode of acaricide treatment adopted by the owners and their experiences about the efficacy of commonly used acaricides. The ticks were collected in separate vials, closed with muslin cloth to allow air and moisture exchange, brought to the laboratory, cleaned, labelled and kept at 28 ± 1 ◦ C and 85 ± 5% relative humidity. Acaricide Technical grade 100% pure cypermethrin (AccuStandard® Inc., USA) was used to prepare the stock solution in methanol (100 mg/ml). For the experimental bioassay, different concentrations of the acaricide were prepared in distilled water from the stock solution and tested against field samples of R. (B.) microplus and H. anatolicum. Adult immersion test The adult immersion test (AIT) was conducted according to the methods of Drummond et al. (1973) and Sharma et al. (2012). Briefly, preweighed engorged females of R. (B.) microplus and H. anatolicum were immersed in different concentrations of cypermethrin (100, 200, 300, 400, and 500 ppm) for 2 min and then soaked in filter paper before transferring them into Petri dishes. After 24 h, ticks were transferred to glass tubes covered with muslin cloth and kept in desiccators kept in a BOD incubator maintained at 28 ± 1 ◦ C and 85 ± 5% RH. Ticks which did not oviposit even after 14 days post treatment were considered dead. The control group was treated in a similar manner in distilled water. Each concentration was replicated twice, and 10 adults were used per replication, and the following parameters were compared:
et al., 2009). The LC50 values of cypermethrin were 138.5 and 245.9 ppm against acaricide-susceptible reference IVRI-I line of R. (B.) microplus (Sharma et al., 2012) and acaricide-susceptible reference IVRI-II line of H. anatolicum (Shyma et al., 2012), respectively, both maintained in the Entomology Laboratory, Indian Veterinary Research Institute, for the past 15 years and not been exposed to any acaricides. On the basis of RF, the resistance status in the field populations of R. (B.) microplus and H. anatolicum was classified as susceptible (RF < 1.4), level I (RF = 1.5–5.0), level II (RF = 5.1–25.0), level III (RF = 25.1–40), and level IV (RF > 40.1) (Sharma et al., 2012). Results In the present study, areas with mixed infestations of R. (B.) microplus and H. anatolicum under similar environmental conditions were covered. Farmers of these areas reported frequent applications of available acaricides particularly cypermethrin without maintaining an optimum concentration for the control of ticks mainly due to low efficacy of most of the marketed products. Hence, uniform treatment was being adopted for the control of both singleand multi-host tick species by the farmers. Cypermethrin resistance status in Muktsar tick samples Data on the LC50 values of cypermethrin, RF values, and the level of resistance to cypermethrin in the Muktsar samples of R. (B.) microplus and H. anatolicum are shown in Table 1. The RF value of R. (B.) microplus was recorded as 5.48, a level II resistance status, whereas H. anatolicum (1.12) had a susceptible status indicating that resistance in the multi-host tick species develops slower. The regression graphs of probit mortality of both tick species plotted against log values of progressively increasing concentrations of cypermethrin are shown in Fig. 1. The egg mass weights produced by both tick species upon exposure with different concentrations of cypermethrin are shown in Fig. 2. The slope of the egg mass weight of R. (B.) microplus (−0.53 ± 0.03) was higher than in H. anatolicum (−1.02 ± 0.13). The R2 values of the egg mass weights of R. (B.) microplus and H. anatolicum were 0.983 and 0.949, respectively (Table 1). The comparative reproductive index (RI) of both tick species is shown in Fig. 3. The slope of RI of R. (B.) microplus was higher than in H. anatolicum showing a susceptible resistance status. The slope and R2 values of R. (B.) microplus ticks for RI was recorded as −0.28 ± 0.05 and 0.912, respectively. In case of H. anatolicum, the respective values were −0.37 ± 0.01 and 0.996, respectively. The comparative percentage inhibition of oviposition (%IO) in these tick species are shown in Fig. 4. The slope of %IO of R. (B.) microplus (57.99 ± 10.38) was lower than in H. anatolicum (61.86 ± 2.04), and the respective R2 values were 0.912 and 0.996, respectively.
Mortality: recorded up to 14 days post treatment Egg mass weight laid by the live ticks Reproductive index (RI) = egg mass wt./engorged female wt Percentage inhibition of oviposition (%IO) = [(RI control − RI treated)/RI control × 100].
Dose–response data were analyzed by the probit method (Finney, 1962) using Graph Pad Prism 4 software. The lethal concentration of cypermethrin for 50% of ticks (LC50 values) was determined by applying regression equation analysis to the probittransformed data of mortality. Resistance diagnosis in field samples of ticks Resistance factors (RF) for field tick samples were worked out by the quotient between LC50 of field ticks and LC50 of susceptible lines of R. (B.) microplus and H. anatolicum (Castro-Janer
R.m. H.a.
6 5 4
Probit mortality
(a) (b) (c) (d)
91
3 2 1 0 1.75
2.00
2.25
2.50
2.75
3.00
Log conc Fig. 1. Dose mortality curves after treatment with cypermethrin in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar.
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Table 1 LC50 and 95% confidence limit, resistance factor, and resistance level against cypermethrin of ticks collected from Muktsar, Punjab, India. Tick
Variables
Slope ± SE (95% CL)
R. (B.) microplus
Mortality Egg mass RI %IO
2.49 −0.53 −0.28 57.99
± ± ± ±
H. anatolicum
Mortality Egg mass RI %IO
2.02 −1.02 −0.37 61.86
± ± ± ±
R2
LC50 (ppm) (95% CL)
RF
RL
0.32 (1.45–3.54) 0.03 (−0.66 to −0.41) 0.05 (−0.43 to −0.12) 10.38 (24.97–91.01)
0.950 0.983 0.912 0.912
759.62 (699.5–824.8)
5.48
II
0.48 (0.46–3.57) 0.13 (−1.45 to −0.58) 0.01 (−0.41 to −0.33) 2.04 (55.34–68.38)
0.851 0.949 0.996 0.996
275.63 (248.9–305.1)
1.12
S
1.25
R.m. H.a.
1.00
0.50 0.25 0.00 -0.25
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 2. Egg mass weight in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar after treatment with cypermethrin.
0.6
0.4 0.3 0.2 0.1 0.0 1.75
2.00
2.25
2.50
2.75
3.00
R.m. H.a.
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 4. Inhibition of oviposition (%IO) in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar after treatment with cypermethrin.
R.m. H.a.
0.5
Reproductive index
70 60 50 40 30 20 10 0 -10 -20
Probit mortality
Egg Mass (g)
0.75
Inhibition of Oviposition (%)
R2 , goodness of fit; RF, resistance factor; RL, resistance level.
Log conc
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 1.75
R.m. H.a.
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 3. Reproductive index in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar after treatment with cypermethrin.
Cypermethrin resistance status in Mansa tick samples Data on LC50 , RF values, and the level of resistance to cypermethrin in the Mansa samples of R. (B.) microplus and H. anatolicum are shown in Table 2. Resistance in R. (B.) microplus was on level I with an RF value of 2.18, whereas H. anatolicum showed a much lower RF (0.82) and was designated as susceptible. The regression graphs of probit mortality of both tick species plotted against log values of progressively increasing concentrations of cypermethrin are
Fig. 5. Dose mortality curves after treatment with cypermethrin in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa.
shown in Fig. 5. The egg mass weights produced by both tick species are shown in Fig. 6. Similar to the Muktsar sample, the slope of the egg mass weight of R. (B.) microplus (−0.49 ± 0.06) showing level I resistance status was higher than in H. anatolicum (−1.23 ± 0.12). The R2 values of the egg mass weights of R. (B.) microplus and H. anatolicum were 0.955 and 0.967, respectively (Table 2). RI and %IO of both ticks are shown in Fig. 7 and Fig. 8, respectively. The slope and R2 values of R. (B.) microplus ticks for RI were recorded as −0.19 ± 0.04 and 0.838, respectively, whereas in case of
Table 2 LC50 and 95% confidence limit, resistance factor, and resistance level against cypermethrin of ticks collected from Muktsar, Punjab, India. Tick
Variables
Slope ± SE (95% CL)
R. (B.) microplus
Mortality Egg mass RI %IO
2.72 −0.49 −0.19 40.26
± ± ± ±
H. anatolicum
Mortality Egg mass RI %IO
2.51 −1.23 −0.34 55.48
± ± ± ±
R2 , goodness of fit; RF, resistance factor; RL, resistance level.
R2
LC50 (ppm) (95% CL)
RF
RL
0.12 (2.32–3.12) 0.06 (−0.69 to −0.30) 0.04 (−0.35 to −0.03) 10.22 (7.75–72.77)
0.993 0.955 0.838 0.838
302.22 (280.2–325.9)
2.18
I
0.66 (0.40–4.61) 0.12 (−1.64 to −0.82) 0.04 (−0.48 to −0.19) 7.34 (32.12–78.85)
0.827 0.967 0.950 0.950
203.23 (187.2–220.6)
0.82
S
N.K. Singh et al. / Ticks and Tick-borne Diseases 5 (2014) 90–94
1.25
R.m. H.a.
1.00
Egg Mass (g)
0.75 0.50 0.25 0.00 -0.25
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 6. Egg mass weight in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa after treatment with cypermethrin.
0.6
R.m. H.a.
Reproductive index
0.5 0.4 0.3 0.2 0.1 0.0 1.75
2.00
2.25
2.50
2.75
3.00
Log conc
Inhibition of Oviposition (%)
Fig. 7. Reproductive index in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa after treatment with cypermethrin.
70
R.m. H.a.
60 50 40 30 20 10 0 -10
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 8. Inhibition of oviposition (%IO) in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa after treatment with cypermethrin.
H. anatolicum, the respective values were −0.34 ± 0.04 and 0.950, respectively. The slope of %IO of R. (B.) microplus (40.26 ± 10.22) was lower than in H. anatolicum (55.48 ± 7.34) and the respective R2 values were 0.838 and 0.950, respectively. Discussion Amongst the different vectors, ticks are ranked second to mosquitoes in terms of the numbers of different pathogens transmitted to human and animals (Zhou et al., 2009). Recent studies have shown that R. (B.) microplus and H. anatolicum are the major tick species infesting dairy animals of Punjab state, India (Haque et al., 2011; Singh and Rath, 2013). Synthetic pyrethroids are the most widely and frequently used acaricides to control tick populations. Selection for acaricide resistance in tick populations is a major consequence of using chemical acaricides and is the principal threat to the efficacy of SPs particularly cypermethrin for the control of these vectors. The purpose of the present study was a comparative analysis of the level of cypermethrin resistance persisting in 2 different field samples of R. (B.) microplus and H. anatolicum in Punjab, India.
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The standard bioassay recommended by FAO for testing resistance to acaricides in ticks is the larval packet test (LPT) originally described by Stone and Haydock (1962). However, the LPT takes 5–6 weeks to complete, is a laborious test and requires significant laboratory resources to conduct the test routinely whilst AIT can be conducted with ease, and data can be generated within 2 weeks time. In the current study, AIT was standardized with a 14 days oviposition protocol as by Sharma et al. (2012) in contrast to the 7 days protocol followed elsewhere (Sabatini et al., 2001). An optimized immersion time of 2 min was used as by Kumar et al. (2011). Because age and condition of ticks prior to AIT are probably important as to the variability of results (Jonsson et al., 2007), these factors were standardized in repeated laboratory experiments, and consistent results were obtained. In the AIT bioassay, technical grade cypermethrin was selected over commercial formulations as the commercial products are prepared with many proprietary ingredients, and it is difficult to assess the responses due to active ingredients (Shaw, 1966). The stock solutions of cypermethrin were prepared by dissolving in 100% methanol, and the working concentrations were prepared with distilled water. Use of suitable organic solvent is an important step in the process of preparation of the various dilutions as it facilitates adsorption of the compound to the surface of target biological materials and also enhances penetration of active ingredients of the acaricide across the exoskeleton (Sharma et al., 2012). In AIT, mortality was used as the main criterion for calculation of LC50 values of both the reference acaricide-susceptible IVRI strains and the field samples of R. (B.) microplus and H. anatolicum because this was the earliest parameter to measure and the chances of data variation was low as the number of ticks used were high. In past studies, absence of repetition in AIT values has been attributed to great data variation in low sample sizes (Jonsson et al., 2007). However, the predictive value of the AIT for acaricide resistance has been improved in the current study by increasing the number of ticks submitted to each trial. Amongst the 2 field samples of R. (B.) microplus and H. anatolicum collected from the same locations of Punjab, India, with uniform usage of cypermethrin, the obtained LC50 values were much lower for the multi-host tick species thus indicating that resistance might slower develop in them. Acaride resistance in ticks is not universal and is most widespread and diverse in one-host cattle ticks of the subgenus Boophilus (Wharton and Roulston, 1970) and has much slower developed in two- and three-host ticks (Rhipicephalus, Amblyomma, Hyalomma, and Ixodes). This is due to the fact that a much larger fraction (every life stage) of the total population of a given one-host tick species than in multi-host ticks is under chemical challenge. In addition to this, a single generation of a multi-host tick species may extend over a much longer period of time up to 3 years compared to 2–3 months in Boophilus (Harley, 1966). Further, the immature stages of multi-host ticks often feed on other hosts, particularly small wild animals, even if the adults tend to prefer large domestic animals. This could be an additional factor reducing exposure to chemicals in these ticks and may have caused a reduction in selection pressure (Kunz and Kemp, 1994). Tick populations have an immense potential for rapidly developing resistance due to their biological and behavioural characteristics, and resistance to different active ingredients has been reported in almost all countries where ticks occur (Alonso-Diaz et al., 2006). A survey based on questionnaires in a sampled population of manufacturers and farmers reported the presence of a widespread acaricidal resistance in India (FAO, 2004). There are several reports available on acaricidal resistance in R. (B.) microplus from various parts of India (Chaudhary and Naithani, 1964; Basu and Haldar, 1997; Singh et al., 2010, 2012). Recently, large-scale
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resistances to the organophosphate compound diazinon (Kumar et al., 2011) and SPs, particularly cypermethrin and deltamethrin (Sharma et al., 2012), have been experimentally validated in Indian samples of R. (B.) microplus. However, there are only few records of SP resistance in multi-host ticks worldwide (Nolan, 1990), particularly H. anatolicum in India (Sangwan et al., 1993; Shyma et al., 2012; Singh et al., 2013), which could be due to a slower development of acaricide resistance in multi-host ticks. The dose–response curves for egg mass weight, the reproductive index, and the %IO of field samples of R. (B.) microplus and H. anatolicum were also validated by AIT. The slope of egg mass weight and the RI of engorged females exposed to various concentrations of cypermethrin were negative thus indicating that although the increase in concentration of the drug could not cause mortality in all the exposed ticks, the egg laying capacity or the efficacy of conversion of live weight into egg mass decreased among the surviving females. This phenomenon was more marked in H. anatolicum than in R. (B.) microplus ticks, which further validates their susceptible status and their slower rate of resistance development against cypermethrin. Recent reports on the prevalence of ixodid ticks have shown R. (B.) microplus as the predominant tick of dairy animals in India, particularly in Punjab state (Ghai et al., 2008; Haque et al., 2011; Singh and Rath, 2013). However, a much earlier report from the state showed that H. anatolicum was the most frequent tick species of cattle in this area (Gill and Gill, 1977). The decline in the populations of multi-host ticks is mainly due to the adaptation of newer and effective tick control measures particularly the usage of chemical acaricides in the past 2–3 decades (Singh and Rath, 2013). Results of the current study further validate the fact that the rate of development of resistance against the commonly used acaricides in multi-host tick species is slower thus maintaining their susceptible status for a much longer period of time which indeed plays a significant role in the changing population dynamics of the ticks in the region. Further in R. (B.) microplus, the phenomenon of early development of resistance against commonly used chemical acaricides and thus rendering them ineffective has largely contributed to making it the predominant tick of dairy animals and overpowering the multi-host tick H. anatolicum. Thus, the data generated on the comparative resistance status in 2 economically important tick species of India might be of immense help in formulating suitable control measures against ticks and tickborne diseases of animals. Acknowledgement The authors are thankful to The Director of Research, Guru Angad Dev, Veterinary and Animal Sciences University, Ludhiana, for providing facilities to carry out the research work. References Alonso-Diaz, M.A., Rodriguez-Vivas, R.I., Fragoso-Sanchez, H., Rosario-Cruz, R., 2006. Resistance of the Boophilus microplus tick to ixodicides. Arch. Med. Vet. 38, 105–114 (in Spanish). Ansari, M.A., Razdan, R.K., 2003. Bio-efficacy and operational feasibility of alphacypermethrin (Fendona) impregnated mosquito nets to control rural malaria in northern India. J. Vector Borne Dis. 40, 33–42. Basu, A., Haldar, D.P., 1997. A note on the effect of continuous use of Sevin 50 WP on some cattle ticks. J. Vet. Parasitol. 11, 183–184. Biswas, S., 2003. Role of veterinarians in the care and management during harvest of skin in livestock species. In: Proceedings of National Seminar on Leather Industry in Today’s Perspective, Kolkata, India, pp. 62–64. Castro-Janer, E., Rifran, L., Piaggio, J., Gil, A., Miller, R.J., Schumaker, T.T.S., 2009. In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their characterization. Vet. Parasitol. 162, 120–128. Chaudhary, R.P., Naithani, R.C., 1964. Resistance to BHC in the cattle tick Boophilus microplus in India. Bull. Entomol. Res. 55, 405–410.
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Ixodid ticks of domestic animals in the Punjab State. PAU, Ludhiana, pp. 2–14. Haque, M., Jyoti, Singh, N.K., Rath, S.S., Ghosh, S., 2011. Epidemiology and seasonal dynamics of ixodid ticks of dairy animals of Punjab state, India. Indian J. Anim. Sci. 81, 661–664. Harley, K.L.S., 1966. Studies on the survival of the nonparasitic stages of the cattle tick Boophilus microplus in three climatically dissimilar districts of North Queensland. Aust. J. Agric. Res. 17, 387–410. Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129, S3–S14. Jonsson, N.N., Miller, R.J., Robertson, J.L., 2007. Critical evaluation of the modifiedadult immersion test with discriminating dose bioassay for Boophilus microplus using American and Australian isolates. Vet. Parasitol. 146, 307–315. Kumar, A., Mahour, K., Gupta, V.K., Vihan, V.S., 2006. Susceptibility and relative resistance in tick population against cypermethrin in organized farm and field animals. Vet. Pract. 7, 41–43. 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Effect of Hyalomma ticks (Acari: Ixodidae) on milk production of dairy buffaloes (Bos bubalus bubalis) of Punjab (Pakistan). Ital. J. Anim. Sci. 6, 939–941. Sangwan, A.K., Chhabra, M.B., Singh, S., 1993. Acaricide resistance status of common livestock ticks of Haryana. Indian Vet. J. 70, 20–24. Sharma, A.K., Kumar, R., Kumar, S., Nagar, G., Singh, N.K., Rawat, S.S., Dhakadd, M.L., Rawat, A.K.S., Ray, D.D., Ghosh, S., 2012. Deltamethrin and cypermethrin resistance status of Rhipicephalus (Boophilus) microplus collected from six agroclimatic regions of India. Vet. Parasitol. 188, 337–345. Sharma, S.N., Saxena, V.K., Lal, S., 2004. Study on susceptibility status in aquatic and adult stages of Aedes aegypti and Aedes albopictus against insecticides at international airports of South India. J. Commun. Dis. 36, 177–181. Shaw, R.D., 1966. Culture of an organophosphorus resistant strain of Boophilus microplus (Canestrini) and assessment of its resistance spectrum. Bull. Entomol. Res. 56, 398–405. Shyma, K.P., Kumar, S., Sharma, A.K., Ray, D.D., Ghosh, S., 2012. Acaricide resistance status in Indian isolates of Hyalomma anatolicum. Exp. Appl. Acarol. 58, 471–481. Singh, N.K., Haque, M., Jyoti, Rath, S.S., 2012. Deltamethrin resistance in Rhipicephalus microplus in Ludhiana. Indian Vet. J. 89, 23–25. Singh, N.K., Jyoti, Haque, M., Rath, S.S., 2010. Studies on acaricide resistance in Rhipicephalus (Boophilus) microplus against synthetic pyrethroids by adult immersion test with a discriminating dose. J. Vet. Parasitol. 24, 207–208. Singh, N.K., Jyoti, Rath, S.S., 2013. Detection of acaricidal resistance in Hyalomma anatolicum anatolicum. Indian Vet. J. 90, 17–19. Singh, N.K., Rath, S.S., 2013. Epidemiology of ixodid ticks in cattle population of various agro-climatic zones of Punjab. Asian Pac. J. Trop. Med. 6, 947–951. Solis, S.S., 1991. Boophilus ticks ecology: perspectives of a panorama. In: Proceedings of II International Seminar on Animal Parasitology on Tick-borne Diseases, Morelos, Mexico, pp. 19–30. Stone, B.F., Haydock, P., 1962. A method for measuring the acaricide susceptibility of the cattle tick Boophilus microplus (Can.). Bull. Entomol. Res. 53, 563–578. Sutherst, R.W., 1983. Management of Arthropod Parasitism in Livestock. World Association for the Advancement of Veterinary Parasitology, Dansmore, Australia. Tiwari, S., Ghosh, S.K., Ojha, V.P., Dash, A.P., Kamaraju, R., 2010. Reduced susceptibility to selected synthetic pyrethroids in urban malaria vector Anopheles stephensi: a case study in Mangalore city, South India. Malaria J. 9, 179. Wharton, R.H., Roulston, W.J., 1970. Resistance of ticks to chemicals. Annu. Rev. Entomol. 15, 381–404. Zhou, J., Liao, M., Ueda, M., Gong, H., Xuan, X., Fujisaki, K., 2009. Characterization of an intracellular cystatin homolog from the tick Haemaphysalis longicornis. Vet. Parasitol. 160, 180–183.
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
A comparative study on cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum from Punjab (India) Nirbhay K. Singh a,∗ , Jyoti a , Manjurul Haque a , Harkirat Singh a , Shitanshu S. Rath a , Srikant Ghosh b a Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141004, India b Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, UP, India
a r t i c l e
i n f o
Article history: Received 11 March 2013 Received in revised form 26 August 2013 Accepted 26 August 2013 Available online 16 November 2013 Keywords: Cypermethrin Hyalomma anatolicum Punjab Resistance Rhipicephalus (Boophilus) microplus
a b s t r a c t A study to evaluate cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Muktsar and Mansa districts of Punjab state, India, was conducted by using adult immersion test (AIT). The regression graphs of probit mortality of ticks plotted against log values of concentrations of cypermethrin was utilized for the determination of slope of mortality, lethal concentration for 50% (LC50 ), and the resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index, and percentage inhibition of oviposition), the resistance levels were categorized. Resistance to cypermethrin was categorized as level II and I in R. (B.) microplus collected from Muktsar and Mansa districts, respectively, whereas, H. anatolicum from both locations showed a susceptible status. The RF values of Muktsar and Mansa field samples of engorged R. (B.) microplus (5.48 and 2.18, respectively) were much higher as those of engorged H. anatolicum (1.12 and 0.82, respectively) indicating a lower level and slower rate of development of cypermethrin resistance in multi-host ticks. The data generated in the current study might be of immense help in formulating suitable control measures against ticks and tick-borne diseases of animals. © 2013 Elsevier GmbH. All rights reserved.
Introduction Ticks and tick-borne diseases are a major problem to livestock health worldwide. Their significance depends on the region, the species involved, the host population(s) involved, and socioeconomic and technological advances in control measures (Solis, 1991). Losses attributable to ticks are caused either directly by tick bites (Biswas, 2003; Jongejan and Uilenberg, 2004), blood loss, damage to hides and udders, decreased milk yield (Sutherst, 1983; Sajid et al., 2007), or indirectly through mortality or morbidity caused by the pathogens transmitted by or associated with the ticks. The global economic loss due to tick infestation has been estimated as US$14,000–18,000 million annually, and the cost of management of ticks and tick-borne diseases in livestock of India is as high as US$498.7 million per annum (Minjauw and Mc Leod, 2003). Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum are widely prevalent and considered as economically important ixodid ticks infesting dairy animals in India (Ghosh et al., 2007), particularly in Punjab state (Haque et al., 2011; Singh and Rath, 2013).
∗ Corresponding author. Tel.: +91 9417336332; fax: +91 1612400822. E-mail address: [email protected] (N.K. Singh). 1877-959X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ttbdis.2013.08.002
Presently, the use of chemical acaricides is the most widely employed tick control strategy in India. These chemicals are easily available and are applied on infested animals at frequent intervals leading to their indiscriminate use which has probably contributed to the development of acaricide resistance in ticks (Sharma et al., 2012). Synthetic pyrethroids (SPs), particularly cypermethrin, is the predominantly used commercially available acaricide employed for tick control in Punjab state, India. Beside its application against agriculturally important pests, it is also used extensively for the control of mosquitoes (Ansari and Razdan, 2003; Sharma et al., 2004; Tiwari et al., 2010). Although cattle owners have reported treatment inefficiency in field conditions, only limited data on tick resistance to cypermethrin are currently available from India (Kumar et al., 2006; Sharma et al., 2012; Shyma et al., 2012) and particularly from Punjab state (Singh et al., 2010, 2013). Further, most of the published data available are regarding the resistance status of R. (B.) microplus, whereas, reports on the resistance status of H. anatolicum are scarce, and no data are available on a comparative basis of the resistance status of single-host versus multi-host ticks. Having in mind the variation in the development of acaricide resistance in different tick types, it becomes essential to generate data for these different tick species for formulation and implementation of effective tick control strategies particularly for countries like India where mixed tick
N.K. Singh et al. / Ticks and Tick-borne Diseases 5 (2014) 90–94
infestation is a common feature. The current study was undertaken to generate data on the cypermethrin resistance status of R. (B.) microplus and H. anatolicum collected from Punjab state, India. Materials and methods Study area Live engorged adult female R. (B.) microplus and H. anatolicum ticks were collected from cross-bred dairy animals with mixed infestation of both tick species as well as their sheds, from Muktsar and Mansa districts located at the trans-gangetic plain in the state of Punjab (29.30◦ N to 32.32◦ N and 73.55◦ E to 76.50◦ E) with humid subtropical climatic conditions. Besides, uniform tick infestation patterns, easy accessibility promoted the selection of these regions. Both organized and unorganized farms were selected to collect the samples. A questionnaire was formulated to collect data on frequency, type, and mode of acaricide treatment adopted by the owners and their experiences about the efficacy of commonly used acaricides. The ticks were collected in separate vials, closed with muslin cloth to allow air and moisture exchange, brought to the laboratory, cleaned, labelled and kept at 28 ± 1 ◦ C and 85 ± 5% relative humidity. Acaricide Technical grade 100% pure cypermethrin (AccuStandard® Inc., USA) was used to prepare the stock solution in methanol (100 mg/ml). For the experimental bioassay, different concentrations of the acaricide were prepared in distilled water from the stock solution and tested against field samples of R. (B.) microplus and H. anatolicum. Adult immersion test The adult immersion test (AIT) was conducted according to the methods of Drummond et al. (1973) and Sharma et al. (2012). Briefly, preweighed engorged females of R. (B.) microplus and H. anatolicum were immersed in different concentrations of cypermethrin (100, 200, 300, 400, and 500 ppm) for 2 min and then soaked in filter paper before transferring them into Petri dishes. After 24 h, ticks were transferred to glass tubes covered with muslin cloth and kept in desiccators kept in a BOD incubator maintained at 28 ± 1 ◦ C and 85 ± 5% RH. Ticks which did not oviposit even after 14 days post treatment were considered dead. The control group was treated in a similar manner in distilled water. Each concentration was replicated twice, and 10 adults were used per replication, and the following parameters were compared:
et al., 2009). The LC50 values of cypermethrin were 138.5 and 245.9 ppm against acaricide-susceptible reference IVRI-I line of R. (B.) microplus (Sharma et al., 2012) and acaricide-susceptible reference IVRI-II line of H. anatolicum (Shyma et al., 2012), respectively, both maintained in the Entomology Laboratory, Indian Veterinary Research Institute, for the past 15 years and not been exposed to any acaricides. On the basis of RF, the resistance status in the field populations of R. (B.) microplus and H. anatolicum was classified as susceptible (RF < 1.4), level I (RF = 1.5–5.0), level II (RF = 5.1–25.0), level III (RF = 25.1–40), and level IV (RF > 40.1) (Sharma et al., 2012). Results In the present study, areas with mixed infestations of R. (B.) microplus and H. anatolicum under similar environmental conditions were covered. Farmers of these areas reported frequent applications of available acaricides particularly cypermethrin without maintaining an optimum concentration for the control of ticks mainly due to low efficacy of most of the marketed products. Hence, uniform treatment was being adopted for the control of both singleand multi-host tick species by the farmers. Cypermethrin resistance status in Muktsar tick samples Data on the LC50 values of cypermethrin, RF values, and the level of resistance to cypermethrin in the Muktsar samples of R. (B.) microplus and H. anatolicum are shown in Table 1. The RF value of R. (B.) microplus was recorded as 5.48, a level II resistance status, whereas H. anatolicum (1.12) had a susceptible status indicating that resistance in the multi-host tick species develops slower. The regression graphs of probit mortality of both tick species plotted against log values of progressively increasing concentrations of cypermethrin are shown in Fig. 1. The egg mass weights produced by both tick species upon exposure with different concentrations of cypermethrin are shown in Fig. 2. The slope of the egg mass weight of R. (B.) microplus (−0.53 ± 0.03) was higher than in H. anatolicum (−1.02 ± 0.13). The R2 values of the egg mass weights of R. (B.) microplus and H. anatolicum were 0.983 and 0.949, respectively (Table 1). The comparative reproductive index (RI) of both tick species is shown in Fig. 3. The slope of RI of R. (B.) microplus was higher than in H. anatolicum showing a susceptible resistance status. The slope and R2 values of R. (B.) microplus ticks for RI was recorded as −0.28 ± 0.05 and 0.912, respectively. In case of H. anatolicum, the respective values were −0.37 ± 0.01 and 0.996, respectively. The comparative percentage inhibition of oviposition (%IO) in these tick species are shown in Fig. 4. The slope of %IO of R. (B.) microplus (57.99 ± 10.38) was lower than in H. anatolicum (61.86 ± 2.04), and the respective R2 values were 0.912 and 0.996, respectively.
Mortality: recorded up to 14 days post treatment Egg mass weight laid by the live ticks Reproductive index (RI) = egg mass wt./engorged female wt Percentage inhibition of oviposition (%IO) = [(RI control − RI treated)/RI control × 100].
Dose–response data were analyzed by the probit method (Finney, 1962) using Graph Pad Prism 4 software. The lethal concentration of cypermethrin for 50% of ticks (LC50 values) was determined by applying regression equation analysis to the probittransformed data of mortality. Resistance diagnosis in field samples of ticks Resistance factors (RF) for field tick samples were worked out by the quotient between LC50 of field ticks and LC50 of susceptible lines of R. (B.) microplus and H. anatolicum (Castro-Janer
R.m. H.a.
6 5 4
Probit mortality
(a) (b) (c) (d)
91
3 2 1 0 1.75
2.00
2.25
2.50
2.75
3.00
Log conc Fig. 1. Dose mortality curves after treatment with cypermethrin in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar.
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Table 1 LC50 and 95% confidence limit, resistance factor, and resistance level against cypermethrin of ticks collected from Muktsar, Punjab, India. Tick
Variables
Slope ± SE (95% CL)
R. (B.) microplus
Mortality Egg mass RI %IO
2.49 −0.53 −0.28 57.99
± ± ± ±
H. anatolicum
Mortality Egg mass RI %IO
2.02 −1.02 −0.37 61.86
± ± ± ±
R2
LC50 (ppm) (95% CL)
RF
RL
0.32 (1.45–3.54) 0.03 (−0.66 to −0.41) 0.05 (−0.43 to −0.12) 10.38 (24.97–91.01)
0.950 0.983 0.912 0.912
759.62 (699.5–824.8)
5.48
II
0.48 (0.46–3.57) 0.13 (−1.45 to −0.58) 0.01 (−0.41 to −0.33) 2.04 (55.34–68.38)
0.851 0.949 0.996 0.996
275.63 (248.9–305.1)
1.12
S
1.25
R.m. H.a.
1.00
0.50 0.25 0.00 -0.25
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 2. Egg mass weight in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar after treatment with cypermethrin.
0.6
0.4 0.3 0.2 0.1 0.0 1.75
2.00
2.25
2.50
2.75
3.00
R.m. H.a.
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 4. Inhibition of oviposition (%IO) in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar after treatment with cypermethrin.
R.m. H.a.
0.5
Reproductive index
70 60 50 40 30 20 10 0 -10 -20
Probit mortality
Egg Mass (g)
0.75
Inhibition of Oviposition (%)
R2 , goodness of fit; RF, resistance factor; RL, resistance level.
Log conc
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 1.75
R.m. H.a.
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 3. Reproductive index in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Muktsar after treatment with cypermethrin.
Cypermethrin resistance status in Mansa tick samples Data on LC50 , RF values, and the level of resistance to cypermethrin in the Mansa samples of R. (B.) microplus and H. anatolicum are shown in Table 2. Resistance in R. (B.) microplus was on level I with an RF value of 2.18, whereas H. anatolicum showed a much lower RF (0.82) and was designated as susceptible. The regression graphs of probit mortality of both tick species plotted against log values of progressively increasing concentrations of cypermethrin are
Fig. 5. Dose mortality curves after treatment with cypermethrin in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa.
shown in Fig. 5. The egg mass weights produced by both tick species are shown in Fig. 6. Similar to the Muktsar sample, the slope of the egg mass weight of R. (B.) microplus (−0.49 ± 0.06) showing level I resistance status was higher than in H. anatolicum (−1.23 ± 0.12). The R2 values of the egg mass weights of R. (B.) microplus and H. anatolicum were 0.955 and 0.967, respectively (Table 2). RI and %IO of both ticks are shown in Fig. 7 and Fig. 8, respectively. The slope and R2 values of R. (B.) microplus ticks for RI were recorded as −0.19 ± 0.04 and 0.838, respectively, whereas in case of
Table 2 LC50 and 95% confidence limit, resistance factor, and resistance level against cypermethrin of ticks collected from Muktsar, Punjab, India. Tick
Variables
Slope ± SE (95% CL)
R. (B.) microplus
Mortality Egg mass RI %IO
2.72 −0.49 −0.19 40.26
± ± ± ±
H. anatolicum
Mortality Egg mass RI %IO
2.51 −1.23 −0.34 55.48
± ± ± ±
R2 , goodness of fit; RF, resistance factor; RL, resistance level.
R2
LC50 (ppm) (95% CL)
RF
RL
0.12 (2.32–3.12) 0.06 (−0.69 to −0.30) 0.04 (−0.35 to −0.03) 10.22 (7.75–72.77)
0.993 0.955 0.838 0.838
302.22 (280.2–325.9)
2.18
I
0.66 (0.40–4.61) 0.12 (−1.64 to −0.82) 0.04 (−0.48 to −0.19) 7.34 (32.12–78.85)
0.827 0.967 0.950 0.950
203.23 (187.2–220.6)
0.82
S
N.K. Singh et al. / Ticks and Tick-borne Diseases 5 (2014) 90–94
1.25
R.m. H.a.
1.00
Egg Mass (g)
0.75 0.50 0.25 0.00 -0.25
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 6. Egg mass weight in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa after treatment with cypermethrin.
0.6
R.m. H.a.
Reproductive index
0.5 0.4 0.3 0.2 0.1 0.0 1.75
2.00
2.25
2.50
2.75
3.00
Log conc
Inhibition of Oviposition (%)
Fig. 7. Reproductive index in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa after treatment with cypermethrin.
70
R.m. H.a.
60 50 40 30 20 10 0 -10
2.00
2.25
2.50
2.75
3.00
Log conc
Fig. 8. Inhibition of oviposition (%IO) in samples of H. anatolicum (H. a.) and R. (B.) microplus (R. m.) from Mansa after treatment with cypermethrin.
H. anatolicum, the respective values were −0.34 ± 0.04 and 0.950, respectively. The slope of %IO of R. (B.) microplus (40.26 ± 10.22) was lower than in H. anatolicum (55.48 ± 7.34) and the respective R2 values were 0.838 and 0.950, respectively. Discussion Amongst the different vectors, ticks are ranked second to mosquitoes in terms of the numbers of different pathogens transmitted to human and animals (Zhou et al., 2009). Recent studies have shown that R. (B.) microplus and H. anatolicum are the major tick species infesting dairy animals of Punjab state, India (Haque et al., 2011; Singh and Rath, 2013). Synthetic pyrethroids are the most widely and frequently used acaricides to control tick populations. Selection for acaricide resistance in tick populations is a major consequence of using chemical acaricides and is the principal threat to the efficacy of SPs particularly cypermethrin for the control of these vectors. The purpose of the present study was a comparative analysis of the level of cypermethrin resistance persisting in 2 different field samples of R. (B.) microplus and H. anatolicum in Punjab, India.
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The standard bioassay recommended by FAO for testing resistance to acaricides in ticks is the larval packet test (LPT) originally described by Stone and Haydock (1962). However, the LPT takes 5–6 weeks to complete, is a laborious test and requires significant laboratory resources to conduct the test routinely whilst AIT can be conducted with ease, and data can be generated within 2 weeks time. In the current study, AIT was standardized with a 14 days oviposition protocol as by Sharma et al. (2012) in contrast to the 7 days protocol followed elsewhere (Sabatini et al., 2001). An optimized immersion time of 2 min was used as by Kumar et al. (2011). Because age and condition of ticks prior to AIT are probably important as to the variability of results (Jonsson et al., 2007), these factors were standardized in repeated laboratory experiments, and consistent results were obtained. In the AIT bioassay, technical grade cypermethrin was selected over commercial formulations as the commercial products are prepared with many proprietary ingredients, and it is difficult to assess the responses due to active ingredients (Shaw, 1966). The stock solutions of cypermethrin were prepared by dissolving in 100% methanol, and the working concentrations were prepared with distilled water. Use of suitable organic solvent is an important step in the process of preparation of the various dilutions as it facilitates adsorption of the compound to the surface of target biological materials and also enhances penetration of active ingredients of the acaricide across the exoskeleton (Sharma et al., 2012). In AIT, mortality was used as the main criterion for calculation of LC50 values of both the reference acaricide-susceptible IVRI strains and the field samples of R. (B.) microplus and H. anatolicum because this was the earliest parameter to measure and the chances of data variation was low as the number of ticks used were high. In past studies, absence of repetition in AIT values has been attributed to great data variation in low sample sizes (Jonsson et al., 2007). However, the predictive value of the AIT for acaricide resistance has been improved in the current study by increasing the number of ticks submitted to each trial. Amongst the 2 field samples of R. (B.) microplus and H. anatolicum collected from the same locations of Punjab, India, with uniform usage of cypermethrin, the obtained LC50 values were much lower for the multi-host tick species thus indicating that resistance might slower develop in them. Acaride resistance in ticks is not universal and is most widespread and diverse in one-host cattle ticks of the subgenus Boophilus (Wharton and Roulston, 1970) and has much slower developed in two- and three-host ticks (Rhipicephalus, Amblyomma, Hyalomma, and Ixodes). This is due to the fact that a much larger fraction (every life stage) of the total population of a given one-host tick species than in multi-host ticks is under chemical challenge. In addition to this, a single generation of a multi-host tick species may extend over a much longer period of time up to 3 years compared to 2–3 months in Boophilus (Harley, 1966). Further, the immature stages of multi-host ticks often feed on other hosts, particularly small wild animals, even if the adults tend to prefer large domestic animals. This could be an additional factor reducing exposure to chemicals in these ticks and may have caused a reduction in selection pressure (Kunz and Kemp, 1994). Tick populations have an immense potential for rapidly developing resistance due to their biological and behavioural characteristics, and resistance to different active ingredients has been reported in almost all countries where ticks occur (Alonso-Diaz et al., 2006). A survey based on questionnaires in a sampled population of manufacturers and farmers reported the presence of a widespread acaricidal resistance in India (FAO, 2004). There are several reports available on acaricidal resistance in R. (B.) microplus from various parts of India (Chaudhary and Naithani, 1964; Basu and Haldar, 1997; Singh et al., 2010, 2012). Recently, large-scale
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resistances to the organophosphate compound diazinon (Kumar et al., 2011) and SPs, particularly cypermethrin and deltamethrin (Sharma et al., 2012), have been experimentally validated in Indian samples of R. (B.) microplus. However, there are only few records of SP resistance in multi-host ticks worldwide (Nolan, 1990), particularly H. anatolicum in India (Sangwan et al., 1993; Shyma et al., 2012; Singh et al., 2013), which could be due to a slower development of acaricide resistance in multi-host ticks. The dose–response curves for egg mass weight, the reproductive index, and the %IO of field samples of R. (B.) microplus and H. anatolicum were also validated by AIT. The slope of egg mass weight and the RI of engorged females exposed to various concentrations of cypermethrin were negative thus indicating that although the increase in concentration of the drug could not cause mortality in all the exposed ticks, the egg laying capacity or the efficacy of conversion of live weight into egg mass decreased among the surviving females. This phenomenon was more marked in H. anatolicum than in R. (B.) microplus ticks, which further validates their susceptible status and their slower rate of resistance development against cypermethrin. Recent reports on the prevalence of ixodid ticks have shown R. (B.) microplus as the predominant tick of dairy animals in India, particularly in Punjab state (Ghai et al., 2008; Haque et al., 2011; Singh and Rath, 2013). However, a much earlier report from the state showed that H. anatolicum was the most frequent tick species of cattle in this area (Gill and Gill, 1977). The decline in the populations of multi-host ticks is mainly due to the adaptation of newer and effective tick control measures particularly the usage of chemical acaricides in the past 2–3 decades (Singh and Rath, 2013). Results of the current study further validate the fact that the rate of development of resistance against the commonly used acaricides in multi-host tick species is slower thus maintaining their susceptible status for a much longer period of time which indeed plays a significant role in the changing population dynamics of the ticks in the region. Further in R. (B.) microplus, the phenomenon of early development of resistance against commonly used chemical acaricides and thus rendering them ineffective has largely contributed to making it the predominant tick of dairy animals and overpowering the multi-host tick H. anatolicum. Thus, the data generated on the comparative resistance status in 2 economically important tick species of India might be of immense help in formulating suitable control measures against ticks and tickborne diseases of animals. Acknowledgement The authors are thankful to The Director of Research, Guru Angad Dev, Veterinary and Animal Sciences University, Ludhiana, for providing facilities to carry out the research work. References Alonso-Diaz, M.A., Rodriguez-Vivas, R.I., Fragoso-Sanchez, H., Rosario-Cruz, R., 2006. Resistance of the Boophilus microplus tick to ixodicides. Arch. Med. Vet. 38, 105–114 (in Spanish). Ansari, M.A., Razdan, R.K., 2003. Bio-efficacy and operational feasibility of alphacypermethrin (Fendona) impregnated mosquito nets to control rural malaria in northern India. J. Vector Borne Dis. 40, 33–42. Basu, A., Haldar, D.P., 1997. A note on the effect of continuous use of Sevin 50 WP on some cattle ticks. J. Vet. Parasitol. 11, 183–184. Biswas, S., 2003. Role of veterinarians in the care and management during harvest of skin in livestock species. In: Proceedings of National Seminar on Leather Industry in Today’s Perspective, Kolkata, India, pp. 62–64. Castro-Janer, E., Rifran, L., Piaggio, J., Gil, A., Miller, R.J., Schumaker, T.T.S., 2009. In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their characterization. Vet. Parasitol. 162, 120–128. Chaudhary, R.P., Naithani, R.C., 1964. Resistance to BHC in the cattle tick Boophilus microplus in India. Bull. Entomol. Res. 55, 405–410.
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