Effect of ivermectin, amitraz and fipronil on midgut epithelium and digestive enzyme profile in Rhipicephalus microplus ticks (Acari: Ixodidae).

Blood feeding and digestion are vital physiological activities essential for the survival and reproduction of ticks. Chemical acaricides viz., ivermectin, amitraz and fipronil, are known to act on the central nervous system, resulting in the mortality of ticks. The present study is focused on the effect of these acaricides on the midgut and gut enzymes of Rhipicephalus microplus. The ultra-thin sections of midgut of ivermectin-treated ticks showed irregular basal membrane and ruptured digestive vesicles. Amitraz treatment resulted in a notable decrease in digestive cells with pleats in the basal membrane, while fipronil-exposed ticks exhibited reduced digestive cells, loss of cellular integrity, and disintegration of the basal membrane and muscle layer. The gut tissue homogenate of ivermectin and fipronil treated ticks showed a significant reduction of cathepsin D level, 76.54 ± 3.20 µg/mL and 92.67 ± 3.72 µg/mL, respectively, as compared to the control group (150.0 ± 3.80 µg/mL). The leucine aminopeptidase level (4.27 ± 0.08 units/mL) was significantly decreased in the ivermectin treated ticks compared to other treatment groups. The acid phosphatase activity (29.16 ± 0.67 µmole/min/L) was reduced in the ivermectin treated group whereas, increased activity was observed in the fipronil and amitraz treated groups. All the treatment groups revealed increased alkaline phosphatase levels (17.47-26.72 µmole/min/L). The present finding suggests that in addition to the established mechanism of action of the tested acaricides on the nervous system, the alterations in the cellular profile of digestive cells and enzymes possibly affect the blood digestion process and thus the synthesis of vital proteins which are essential for vitellogenesis, and egg production in ticks.

Evaluation of cytotoxic effects of amitraz and fipronil on digestive, reproductive and neural processes of engorged Rhipicephalus microplus (Acari: Ixodidae) female.

Fipronil and amitraz are potentially toxic compounds used for controlling ticks infesting pet and livestock. The use of fipronil on large animals was limited because of its high costs while amitraz is still persisting in the market since its introduction over four decades ago. Though resistance in ticks against these pesticides has been reported worldwide since 2000, the toxicity of these chemicals at cellular level in ticks is still poorly understood. The present study aimed to examine the gross and cellular impact of fipronil and amitraz on the gut, ovaries and synganglion of engorged Rhipicephalus microplus females. Fipronil and amitraz treated tick groups showed formation of a large number of vacuoles of different size throughout the cytoplasm of generative cells whereas sessile, residual and detached digestive cells were very low in numbers. The treatment of ticks resulted in the formation of vacuolations at periphery of all oocytes. Ultra-thin sections of the synganglion revealed severe rupture of neural lamella and perineurium with apoptosis of neural cells after fipronil treatment whereas in the amitraz treated ticks, severe destruction of neuropile region and extensive vacuolation of type I and II cells of cortical region as compared to the unexposed ticks were noted.

Synergistic property of piperonyl butoxide, diethyl maleate, triphenyl phosphate and verapamil hydrochloride with deltamethrin and ivermectin against Rhipicephalus microplus ticks.

The present study was taken up to evaluate the synergistic properties of piperonyl butoxide (PBO), diethyl maleate (DEM), triphenyl phosphate (TPP) and verapamil (VER) with deltamethrin (DLM) and ivermectin (IVM) against DLM and IVM resistant tick populations collected from Madhya Pradesh and Punjab states of India. The collected field tick populations were resistant to DLM (Resistance Factor [RF] in the range of 21.71-32.98) and IVM (RF in the range of 1.89-4.98). A strong synergism between DLM and, IVM with PBO and IVM with VER was noticed. The synergistic efficacy of PBO and VER with IVM in reducing the lethal concentration 50 (LC50) value (1.69-5.72 times for PBO and 3.00-10.62 times for VER) of IVM in resistant ticks suggest that a combination of these synergists with IVM can significantly enhance the effectiveness of IVM against IVM-resistant Rhipicephlaus microplus populations gradually establishing in the different parts of the country. The synergistic efficiency of PBO with DLM in reducing the LC50 value was 2.65 and 18.01 times, respectively, against DLM- resistant two R. microplus populations (KTN and LDH). The study revealed the gradual establishment of DLM and IVM resistant populations in the surveyed states suggesting the need to adopt required resistance management strategies. The use of synergists with DLM and IVM has emerged as an effective approach for controlling the acaricide-resistant ticks.

Detection of deltamethrin, cypermethrin and flumethrin efficacy against buffalo lice-Haematopinus tuberculatus.

The cattle and buffalo farm practices have been adopted differently by farmers in India but the infestation of ectoparasites including louse has been advocated in high population of animals across the country. The aim of this study was to identify the louse morphologically and determine the in vitro efficacy of the insecticides deltamethrin, cypermethrin and flumethrin against the buffalo louse, Haematopinus tuberculatus. The present research work was conducted using lice collected from organized buffalo dairy farms of Mhow block, Indore district of Madhya Pradesh, India. The adult's lice were collected from heavily infested regions of the body and tail of buffaloes. Some of the collected adult's lice were preserved for morphological identification in 70% alcohol. Briefly, in vitro treated surface bioassay utilizing a cloth rectangle that allows lice to move freely has been used. The concentrations were prepared as 30, 60, 90 and 120 ppm for deltamethrin and flumethrin, whereas for cypermethrin, 100, 200, 300 and 400 ppm concentrations were prepared in distilled water. The 600 µl of each concentration was spread evenly over a cloth rectangle held in the bottom of a Petri plate. Ten adult lice were used for each concentration in triplicate (n = 30) and the same is maintained for control. The vitality of the louse was assessed at various intervals: 30, 60, 120, 180 and 240 min. The lousicidal efficacy was determined by using in vitro bioassays with deltamethrin, cypermethrin and flumethrin. It is observed that as the concentration of insecticides increases with exposure time, mortality of lice is also increased. The current study reveals that cypermethrin and flumethrin were effective in their recommended doses but in the case of deltamethrin, the lice showed a low level of resistance. Furthermore, this type of study on buffalo louse has not conducted in Mhow region of Madhya Pradesh where heavy infestation of lice occurs on buffalo.

Evaluation of acaricidal resistance status of Rhipicephalus microplus ticks from the hilly state (Uttarakhand) of India and evaluation of efficacy of a natural formulation for the management of resistant ticks.

The resistance status against deltamethrin, cypermethrin, coumaphos and ivermectin was assessed of Rhipicephalus microplus from five districts of Uttarakhand, through adult immersion test and larval packet test. The field isolates were highly resistant to deltamethrin (median resistance ratio [RR50] = 9.10-29.13-fold) followed by cypermethrin (2.23-3.55). Surprisingly the isolates were susceptible to coumaphos (0.34-3.17). Emerging resistance against ivermectin (1.55-3.27) was also observed in all the isolates. Elevated levels of esterases (enzyme ratio = 2.93-5.84-fold), glutathione S-transferases (5.10-10.06) and monooxygenases (1.68-4.02) in resistant fields isolates were highly correlated (47.4-86.0%) with the resistant factor (RR50) determined by bioassay. All the isolates except Uttarkashi possess mutation at the 190th position in domain II of the sodium channel gene. As a mitigation strategy an Ageratum conyzoides-based characterized natural formulation was tested against all the isolates and was found effective at the concentration of 10.1-11.5%. The possibility of using the natural formulation for the management of multi-acaricide resistant ticks is discussed.

Comparative susceptibility of Rhipicephalus microplus collected from the northern state of India to coumaphos, malathion, deltamethrin, ivermectin, and fipronil.

The chemical-based tick management method is gradually losing its clutch due to the establishment of resistant ticks. For development of region-specific tick management strategies, the present study was aimed to evaluate the comparative resistance profile of Rhipicephalus microplus isolates collected from seven districts of Uttar Pradesh, a northern state of India. Comparative analysis of the dose-response data using adult immersion test (AIT) against coumaphos, malathion, deltamethrin, ivermectin, and fipronil revealed that all the isolates were resistant to discriminating concentration of deltamethrin having LC50 of 295.12-436.52 ppm with a resistance ratio of 22.02-32.58. An emerging low level of ivermectin resistance (resistance ratio, RR50 = 1.03-2.26) with LC50 in the range of 22.39-48.98 ppm was found across the isolates. The coumaphos was highly effective against all except Amethi (AMT) isolate. Similarly, malathion was efficacious against most of the isolates except Pratapgarh (PRT) and Sultanpur (SUL) isolates showing LC50 of 5128.61 and 5623.41 ppm, respectively. All the isolates were responsive to fipronil. Comparative detoxifying enzymes profiles revealed a significant correlation between the increased activity of esterase and deltamethrin resistance. The GST activity was 51.2% correlated with RR50 of malathion while esterase activity was significantly correlated (68.9%) with RR50 of coumaphos. No correlation between the ivermectin resistance and enzyme activity was established. Multiple sequence analysis of S4-5 linker region of the sodium channel gene of all the isolates revealed a point mutation at 190th position (C190A) which is associated with deltamethrin resistance. The possible tick management strategies in this part of the country are discussed.

Development of an efficient antitick natural formulation for the control of acaricide-resistant ticks on livestock.

Animal production has a key role in global economic development and food security. Ticks, specifically Rhipicephalus microplus cause substantial economic and health impacts on more than eighty percent of the world cattle population. Though synthetic acaricides play a major role in tick management, their injudicious usage has caused environmental pollution and also promote the establishment of multi-acaricide resistant tick populations which is a matter of great concern. To provide an effective tool for controlling these resistant ticks, the present work was aimed to develop safe and inexpensive antitick natural formulations. Our bioprospection studies of Ageratum conyzoides plant established it as a species potentially having strong acaricidal activity due to the presence of potent acaricidal phyto-chemicals. To develop a suitable antitick natural formulation, 41 samples/fractions/formulations were prepared from the dry powder of the whole aerial part of the A. conyzoides plant using different techniques and delivery matrices. The strongest antitick effect was recorded for formulation ACF6, which demonstrated 87 ± 6% mean mortality with 57 % inhibition of oviposition in treated female ticks. Ticks treated with the ACF6 formulation showed a significant (p < 0.001) reduction in cuticular protein (1.238 ± 0.01 mg/mL) as compared to control ticks (2.928 ± 0.01 mg/mL) but no significant difference in chitin content of treated ticks and control ticks was observed. The formulation was found safe in a rat model as no significant differences in biochemical and haematological parameters among treated and control rats were noted. Histopathological studies indicated no sign of hepatocellular necrosis and no significant changes in the weights of liver and spleen was recorded. The overall in vivo efficacy of the formulation was 85 % for experimentally infested cattle with direct mortality of more than 80 % within 96 h post-application. The lethal effect of the formulation was in the form of drying and dead ticks 1-2 d after application. The developed formulation has the potential to be adopted as an alternative tick control measure in an ecofriendly manner.

Molecular tools-advances, opportunities and prospects for the control of parasites of veterinary importance.

The recent advancement in genome sequencing facilities, proteomics, transcriptomics, and metabolomics of eukaryotes have opened door for employment of molecular diagnostic techniques for early detection of parasites and determining target molecules for formulating control strategies. It further leads to the introduction of several purified vaccines in the field of veterinary parasitology. Earlier, the conventional diagnostic methods was entirely based upon morphological taxonomy for diagnosis of parasites but nowadays improved molecular techniques help in phylogenetic study and open an another area of molecular taxonomy of parasites with high precision. Control measures based upon targeting endosymbionts in parasites like Dirofilaria immitis is also under exploration in veterinary parasitology. Metagenomics have added an inside story of parasites bionomics which have created havoc in human and animals population since centuries. Omics era is playing a key role in opening the new approaches on parasite biology. Various newer generations of safer vaccines like edible vaccines and subunit vaccines and diagnostic techniques based upon purified immunologically active epitopes have become commercially available against the parasites (helminths, protozoa and arthropod borne diseases). Nowadays, a transgenic and gene knock out studies using RNA interference and CRISPR are also helping in understanding the functions of genes and screening of target genes, which are not available before the advent of molecular tools. Molecular techniques had paramount impact on increasing the sensitivity of diagnostic tools, epidemiological studies and more importantly in controlling these diseases. This review is about the advancements in veterinary parasitology and their impact on the control of these pathogens.

Association between overexpression of cytochrome P450 genes and deltamethrin resistance in Rhipicephalus microplus.

Cytochrome P450 monooxygenases mediated metabolic detoxification has been recognized as one of the mechanisms involved in resistance to pyrethroids, which is a class of pesticides that includes acaricides such as deltamethrin. Several cytochrome P450 (CYP) genes were identified in arthropod pests which are upregulated in response to exposure to pesticides used as acaricides. However, to date, limited information is available with respect to CYP genes and their response to acaricide exposure in ticks. We cloned and sequenced four CYP genes, the CYP41, CYP3006G8, CYP319A1 and CYP4W1 from reference susceptible IVRI-I strain of Rhipicephalus microplus. The expression pattern of the genes was investigated using qPCR in reference susceptible IVRI-I, pyrethroid-resistant IVRI-IV and multi-acaricide resistant IVRI-V strains. The effect of a single exposure of deltamethrin, at a concentration of 2600 µg/mL and 299.7 µg/mL on IVRI-IV and IVRI-V strains, respectively, on the expression of the four CYP genes was evaluated. In IVRI-IV strain, the CYP41 gene was highly overexpressed (FC 8.72) while CYP3006G8 was underexpressed with FC of 0.06. All the four genes were overexpressed in IVRI-V strain. After exposure to deltamethrin, the CYP3006G8 transcript levels were significantly upregulated at all time intervals in both resistant strains with the highest FC of 11.62 at 12 h in IVRI-IV and 13.38 at 3 h in IVRI-V. Our results suggest that the constitutive overexpression of CYP41 and deltamethrin induced upregulation of CYP3006G8 contribute to the development of pyrethroid resistance, specifically deltamethrin, in these two reference strains.

Standardization of tick specific biochemical tools for estimation of esterases, monooxygenases and glutathione S-transferase for characterization of acaricide resistance.

Protocols to determine metabolic resistance in ticks were mainly derived from reports published using mosquitoes and agriculturally important insects without prior standardization. In the present study, biochemical assays were standardized to quantify acaricide metabolizing enzymes in tick homogenates. Three variables viz., age, number of larvae and reaction time were optimized using reference susceptible IVRI-I and deltamethrin resistant IVRI-IV (Resistance Factor = 194) tick strains. The optimum conditions for estimation of general esterases were 10-15 day old 40 larvae with 15 mins reaction time, 15-20 day old 40 larvae with 20 mins reaction time for Glutathione S- transferase, while 10-15 day old 80 larvae with 5 mins reaction time for monooxygenase. The standardized protocols were further validated in multi acaricide resistant strain (IVRI-V) and in nine field isolates having variable resistant factors to different acaricides. In all the nine heterogeneous field isolates, a significant correlation (p < .05) between resistance to synthetic pyrethroids and over-expression of esterases and monooxygenase was noticed. Similarly, esterases and GST activities were significantly correlated with resistance to organophosphates. The details of the assay protocol are explained for adoption in different laboratories.

Emergence of fipronil resistant Rhipicephalus microplus populations in Indian states.

The intensive usage of chemical acaricides for the control of the cattle tick Rhipicephalus microplus has resulted in the development and establishment of multi-acaricide resistant populations. Fipronil, a phenylpyrazole insecticide, is currently marketed in India for the management of this important veterinary tick species. Here, we tested Indian isolates of R. microplus which have developed multi-acaricide resistance, for their susceptibility to fipronil. Twenty-five field isolates from five agro-climatic zones of the country were collected and tested by adult immersion test (AIT) and larval packet test (LPT). Sixteen isolates with resistance factor (RF) in the range of 1.56-10.9 were detected using LPT, whereas only 11 isolates with RF ranging from 1.05 to 4.1 were detected using AIT. A significant variation of RF between both tests was found, which raises doubt about the suitability of larva-based assays in screening of fipronil resistance. The data indicated possible cross-resistance between groups of acaricides in fipronil-resistant tick populations.

Acaricide resistance in Rhipicephalus microplus collected from selected districts of Madhya Pradesh, Uttar Pradesh and Punjab states of India.

The isolates of Rhipicephalus microplus collected from Madhya Pradesh (MP), Punjab (PJB) and Uttar Pradesh (UP) states of India were characterized using laboratory standardized adult immersion test (AIT) against macrocyclic lactone (ivermectin), synthetic pyrethroids (cypermethrin and deltamethrin), organophosphates (coumaphos and diazinon) and phenylpyrazole compounds (fipronil). Out of the six isolates tested, five isolates except MTH were resistant to deltamethrin and cypermethrin at level II with RF ranging from 16.4 to 24.02 and 7.05 to 13.2, respectively. In case of organophosphates, coumaphos was less effective showing resistance level II (RF 8.52-11.2) in all the six populations compared with diazinon to which three isolates (MHW, RWA and AGS) were resistant at level II. Except MTH, other five isolates were categorized at level I with RF ranging from 1.53 to 3.02 against ivermectin. The phenylpyrazole compound however was found effective, and none of the isolates could survive at a discriminating concentration. The possible strategy for the management of multi-acaricide-resistant ticks in the surveyed districts was discussed in the present study.

Characterization of acaricide resistance in Rhipicephalus microplus populations infesting cattle in northeastern India and assessment of local plant extracts for tick management.

In the present study, the tick isolates were collected from Assam state, of northeastern region (NER) and characterized using in vitro bioassay, biochemical and molecular assays. Comparing LC50 value of susceptible IVRI-I and larvae of field isolates, revealed that RF against deltamethrin was highest for Morigaon (MGN = 21.8) and lowest for Sonitpur (SNP = 3.3) isolate. The RF against cypermethrin was highest for Nagaon (NGO = 5.0) and lowest for Barpeta (BPT = 1.2) isolate. Against coumaphos, the highest RF of 4.5 was calculated for BPT (4.5) and lowest for NGO (1.3) isolate. While using adults based assay, highest RF of 24.68 against deltamethrin and lowest RF of 4.96 was determined for MGN and SNP isolate, respectively. In contrast to the results obtained using larvae, against cypermethrin, highest RF was recorded for Kamrup Metropolitan (KMP) while it was NGO isolate using larvae. In case of coumaphos, both larvae and adults of BPT isolate were also highly resistant and lowest RF was detected in SNP (2.30) isolate. All the isolates were susceptible to ivermectin. A significant correlation (p < 0.01) between deltamethrin resistance and higher expression of glutathioneS-transferase was observed while no correlation with esterase and monooxygenase enzymes activity was noted. For the development of possible ecofriendly control measure, different accessions of Argemone mexicana and Datura metel plant species were collected, extracted and screened against adult ticks. Two accessions, NEA-03 and NED-06 collected from Amlighat and Diphu (East Karbi Anglong) were more than 90 % effective. Further dose response study of these accessions determined the LC50 values of 4.86 and 3.96 %, respectively.The resistance status of the collected tick isolates was compared with the data generated from other regions having higher livestock population and possibility of exploitation of identified plant species for the development of natural antitick product is discussed.

Comparative impact of coumaphos, amitraz and plant extract of Ageratum conyzoides on the oogenesis of Rhipicephalus microplus.

The present experiment was conducted to evaluate and compare the impact of Ageratum conyzoides plant extract (ACE) with routinely used synthetic acaricides i.e., amitraz and coumaphos on the oogenesis of engorged adult females of Rhipicephalus microplus tick. On the day of dropping from the host, panoistic ovary of R. microplus appeared white in colour, horseshoe shaped, hollow tubular organ with immature oocytes predominantly in dorsal groove. Different developmental stages of oocytes (I-V) proceed simultaneously and asynchronously. Oocytes showed gradual increase in size, deep brown colored with accumulation of eggs in oviduct during 24-72 hours of development.At LC90 concentration a highly significant (p < 0.001) cessation of egg laying after exposure to amitraz and ACE while significant reduction (p < 0.01) of egg laying in coumaphos treated ticks was observed. Upon dissection of treated ticks, uterus and oviduct packed with eggs, which failed to pass out was observed. The histo-architectural alterations including presence of extensive vacuolation, alteration of oocyte morphology, deformation of chorion and disorganization of yolk granules were observed in the treated ovaries. Histochemically, low level of storage or synthesis of essential elements viz., proteins, polysaccharides and lipids in treated oocytes responsible for reduction of fertility and inhibition of progress of vitellogenesis was observed.

Development of a recombinant TaSP-based Dot-ELISA for detection of Theileria annulata infection in cattle.

The study was conducted to develop and validate Dot-ELISA for the diagnosis of Theileria annulata infection in cattle using recombinant Theileria annulata surface protein (r-TaSP). The r-TaSP based indirect plate-ELISA was used as a reference test to compare the efficacy of the Dot-ELISA. The Dot-ELISA was optimized with 500 ng of antigen per dot, 1:150 dilution of serum and 1:1000 dilution of secondary antibody for positive and negative reaction. A total of 17 confirmed positive, 25 negative and 129 field sera samples were used to calculate the diagnostic accuracy of Dot-ELISA in comparison with indirect plate-ELISA. The diagnostic sensitivity and specificity of the Dot-ELISA was 95.8 per cent (95% CI, 93.1-97.2) and 80 per cent (95% CI, 48.1-96.2), respectively. The positive predictive value (PPV) of Dot-ELISA was 98.2 percent (95% CI, 95.5-99.7) and negative predictive value (NPV) was 61.6 percent (95% CI, 37-74). The positive and negative likelihood ratios were 4.79 (95% CI, 1.8-25.69) and 0.053 (95% CI 0.03-1.4), respectively. The Dot-ELISA showed moderate agreement (k value, 0.67, 95% CI, 0.36- 0.82) with indirect plate-ELISA. The developed Dot-ELISA is less expensive and convenient for the diagnosis of T. annulata infection in cattle under field conditions.

Establishment of a multi-acaricide resistant reference tick strain (IVRI-V) of Rhipicephalus microplus.

Tick-borne diseases is a global threat and tick resistance to commonly used acaricides is a growing problem, thus calling for improved resistance monitoring tools. To aid in monitoring of resistance in field tick populations, a resistant colony of Rhipicephalus microplus was characterized with the aim to establish a reference multi-acaricide resistant tick strain. Using a standardized adult immersion test, the Lethal Concentration(LC)50 values for deltamethrin, cypermethrin, fenvalerate and diazinon against the laboratory selected resistant tick (LSRT) strain were determined as 306.7 ppm, 2776.9 ppm, 30262.1 ppm and 9458.7 ppm. Relative to the susceptible IVRI-I tick strain, the LSRT strain showed 4.78- and 5.84-fold increases in activity of esterases, a 6-fold increase for monooxygenases and a 2.24 fold increase for glutathione S-transferase. In the acetylcholinesterase 2 gene, 22 single nucleotide polymorphisms (SNPs) were identified in the LSRT strain. Four of these SNPs lead to amino acid substitutions and were consistently found in resistant field populations in India. A C190A mutation in the domain II S4-5 linker region of sodium channel gene resulting in a L64I amino acid substitution was recorded in the LSRT strain. Monitorable indicators for the maintenance of the strain, designated as the reference IVRI-V tick strain and representing the first established multi-acaricide resistant tick strain in India, were identified.

Determination and validation of discriminating concentration of ivermectin against Rhipicephalus microplus.

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 organophosphate (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% mortality of treated ticks were determined to work out discriminating concentration (DC) in order to diagnose resistance in the field situation. The DC (2 x LC95) 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 RR50 and RR95 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 (LC50 = 21.68, LC95 = 46.77 ppm, RR = 1.0). Amongst the field isolates, the isolate collected from Fatehgarh Sahib district (FTG) of Punjab state showed highest RR50 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.

Characterization and establishment of a reference deltamethrin and cypermethrin resistant tick line (IVRI-IV) of Rhipicephalus (Boophilus) microplus.

The problem of ticks and tick borne diseases is a global threat and growing reports of resistance to commonly used insecticides further aggravated the condition and demands for country specific resistance monitoring tools and possible solutions of the problem. Establishment of standard reference is prerequisite for development of monitoring tools. For studying possible role of different mechanisms involved in development of resistance in Rhipicephalus (Boophilus) microplus population and to develop newer drug to manage the problem of resistance, a deltamethrin exposed and selected tick colony, referred to as IVRI-IV, was characterized using reference susceptible IVRI-I tick line as control. The RF values of IVRI-IV ticks against deltamethrin, cypermethrin and diazinon were determined as 194.0, 26.6, 2.86, respectively, against adults. The esterase enzyme ratios of 2.60 and 5.83 was observed using α-naphthyl and ß-naphthyl acetate while glutathione S-transferase (GST) ratio was 3.77. Comparative analysis of IVRI-I and IVRI-IV carboxylesterase gene sequences revealed 13 synonymous and 5 non synonymous mutations, reported for the first time. The C190A mutation in the domain II S4-5 linker region of sodium channel gene leading to leucine to isoleucine (L64I) amino acid substitution was also detected in the IVRI-IV population. In the present study, monitorable indicators for the maintenance of the reference IVRI-IV colony, the first established deltamethrin and cypermethrin resistant tick line of India, were identified.