Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Research paper Determination and validation of discriminating concentration of ivermectin against Rhipicephalus microplus Abhijit Nandi 1 , Sharath V. Sagar, Gajanan Chigure, Ashutosh Fular, Anil Kumar Sharma, Gaurav Nagar, Sachin Kumar, B.C. Saravanan, Srikant Ghosh ⁎ Entomology Laboratory, Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122 (UP), India ARTICLE INFO Keywords: Discriminating concentration Ivermectin Resistance Rhipicephalus microplus ABSTRACT Rhipicephalus microplus, the major cattle tick species of India is prevalent all over the country and causes huge economic loss directly or indirectly to the dairy industries. Chemical acaricides are playing an important role in managing tick infestations on livestock for many years and consequently, resistance to commonly used orga- nophosphate (OP) and synthetic pyrethroid (SP) compounds has been reported. Subsequently, ivermectin (IVM) has been emerged as an alternative to manage OP and SP resistant ticks. However, with the increase of use during the last 5–8 years, there is a possibility of development of resistance and thus there is an urgent need to develop a robust resistance monitoring tool to safeguard the drug. Lethal concentrations for 50 and 95% mor- tality of treated ticks were determined to work out discriminating concentration (DC) in order to diagnose resistance in the field situation. The DC (2 x LC 95 ) was determined as 93.54 ppm using an established reference susceptible IVRI-1 line of R. microplus adopting adult immersion test. For validation of DC, the resistance status was checked in seven tick isolates of R. microplus collected from northern and eastern regions of India. The RR 50 and RR 95 values of the field isolates against ivermectin were determined and were in the range of 1.56–8.25 and 1.93–27.58, respectively. All the collected isolates were found to have higher lethal concentration and resistance ratio in comparison to reference susceptible IVRI-1 tick line (LC 50 = 21.68, LC 95 = 46.77 ppm, RR = 1.0). Amongst the field isolates, the isolate collected from Fatehgarh Sahib district (FTG) of Punjab state showed highest RR 50 of 8.25 indicating high level of resistance to IVM. The generated DC will be used for IVM resistance characterization of ticks infesting cattle in different parts of the country. 1. Introduction The discovery of Ivermectin (IVM) along with penicillin and aspirin has been considered as one of the major events in drug discovery re- search for its versatility, safety and beneficial impact on human and animal health. The IVM was discovered in 1975 and was the first commercially available broad-spectrum anti-parasiticide having ability to kill both internal and external parasites (Campbell, 1981; Putter et al., 1981; Crump and Ōmura, 2011). After extensive research, Merck and Co. introduced IVM and its analogues in the animal health market in 1981 and became world’s largest selling animal health product, with a wide safety margin (Laing et al., 2017). Ivermectin and other macrocyclic lactones are the most commonly used endo-ectocides in the livestock industry, particularly for the con- trol of gastro-intestinal nematodes, lungworm and various ectoparasites including Rhipicephalus microplus, the major cattle tick of tropical and sub-tropical countries including India and cause significant economic loss directly or indirectly to the dairy industry (Ghosh et al., 2007). Different groups of chemical acaricide are in use for many years in tick infested countries for the management of ticks. However, due to in- discriminate and continuous use of the commonly available acaricides, resistance against different classes of acaricides viz. organophosphate (OP), synthetic pyrethroid (SP) and formamidine has been reported from different parts of the world (Li et al., 2003; Jonsson et al., 2007; Miller et al., 2002; Rosado-Aguilar et al., 2008; Raynal et al., 2013; Vudriko et al., 2016) and the reports are fast pouring in the data base. In India, after the establishment of discriminating concentration (DC) of commonly available acaricides, resistance data were enriched with a number of reports from different parts of the country (Kumar et al., 2011, 2014, 2015; Sharma et al., 2012; Singh et al., 2010, 2014; Shyma et al., 2015). It is observed that a number of tick isolates have devel- oped resistance to multiple groups of acaricide (Ghosh et al., 2015). https://doi.org/10.1016/j.vetpar.2017.12.009 Received 25 August 2017; Received in revised form 6 December 2017; Accepted 8 December 2017 ⁎ Corresponding author. 1 Present address: Dept. of Veterinary Parasitology, West Bengal University of Animal and Fisheries Sciences, 37 K.B. Sarani, Belgachia, Kolkata, 700037, West Bengal, India. E-mail address: sghoshtick@gmail.com (S. Ghosh). Veterinary Parasitology 250 (2018) 30–34 0304-4017/ © 2017 Elsevier B.V. All rights reserved. T