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.

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.

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.

Esterase and glutathione S-transferase levels associated with synthetic pyrethroid resistance in Hyalomma anatolicum and Rhipicephalus microplus ticks from Punjab, India.

Larval packet test was used for assessment of resistance status against cypermethrin and deltamethrin in Hyalomma anatolicum and Rhipicephalus microplus from various districts of Punjab (India). Among the various field isolates of H. anatolicum susceptible status was recorded against cypermethrin in all isolates, whereas against deltamethrin resistance status (level I-III) was recorded. In R. microplus lower resistance levels (I-II) were recorded against cypermethrin in comparison to deltamethrin (level I-IV). Quantitative analysis of general esterase activity revealed a range of 4.21 ± 0.46 to 6.05 ± 0.55 and 2.23 ± 0.23 to 2.66 ± 0.24 µmol/min/mg protein for α- and ß-esterase activity, respectively, in different field isolates of H. anatolicum and the increase in comparison to susceptible was not significant (P > 0.05). In contrast to H. anatolicum, the α- and ß-esterase activity in all field isolates (except Jalandhar) of R. microplus was higher (range of 3.89 ± 0.26 to 10.85 ± 0.47 and 1.75 ± 0.08 to 5.87 ± 0.29 µmol/min/mg protein, respectively) (P < 0.001). The glutathione-S-transferase (GST) activity in field isolates of H. anatolicum and R. microplus was in the range of 0.01 ± 0.001 to 0.03 ± 0.001 and 0.02 ± 0.0003 to 0.03 ± 0.001 mM/mg/min. The enzyme ratios (α-and ß-esterase and GST) and RR95 against deltamethrin of H. anatolicum isolates were correlated (P < 0.05), whereas in R. microplus only α-and ß-esterase and RR50 against deltamethrin were correlated (P < 0.05).

Acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus.

Detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids (SPs) against Rhipicephalus (Boophilus) microplus collected from Moga, Punjab (India) was carried out using larval packet test. Results indicated the presence of resistance of level I and III against cypermethrin (resistance factors (RF) = 4.67) and deltamethrin (RF = 34.2), respectively. Adult immersion test was used to assess the acaricidal activity of aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo, and Withania somnifera along with roots of V. negundo against the SP resistant engorged females of R. (B.) microplus. The efficacy of various extracts was assessed by estimation of percent adult mortality, reproductive index (RI), percent inhibition of oviposition (%IO), and hatching rate. A concentration dependent increase in tick mortality was recorded which was more marked with various ethanolic extracts, and highest mortality was recorded in ticks treated with ethanolic extract of leaves of C. winterianus. The LC50 values were determined by applying regression equation analysis to the probit transformed data of mortality for various aqueous and ethanolic extracts. Acaricidal property was recorded to be higher in ethanolic extracts, and high activity was found with the ethanolic extract of leaves of C. winterianus with LC50 (95% CL) values of 0.46% (0.35-0.59%), followed by W. somnifera as 5.21% (4.45-6.09%) and V. negundo as 7.02% (4.58-10.74%). The egg mass weight of the live ticks treated with different concentrations of the various extract was significantly (p < 0.01) lower than that of control ticks; consequently, the RI and the %IO value of the treated ticks were reduced. Further, complete inhibition of hatching was recorded in eggs laid by ticks treated with ethanolic extracts of leaves of V. negundo and aqueous extracts of leaves of W. somnifera. The results of the current study indicate that extracts of C. winterianus, V. negundo, and W. somnifera can be used for the control of SP resistant ticks.

Detection of amitraz resistance in Rhipicephalus (Boophilus) microplus from SBS Nagar, Punjab, India.

The resistance status of Rhipicephalus (Boophilus) microplus collected from SBS Nagar, Punjab, was evaluated against amitraz by Adult Immersion Test (AIT). The regression graph of probit mortality of ticks plotted against log values of progressively increasing concentrations of amitraz revealed the slope of mortality (95% CI) as 2.36 ± 0.61 (0.38 to 4.33). The LC50 (95% CI) and LC95 (95% CI) values were recorded as 332.52 ppm (305.06-362.44) and 1646.93 ppm (1383.97-1959.84), respectively, and the resistance factor (RF) was 13.17 which indicated level II resistance status. The dose response curves for egg mass weight, reproductive index, and percentage inhibition of oviposition were also validated by AIT and the slopes (95% CI) were -7.17 ± 2.41 (-14.85 to 0.50), -0.009 ± 0.02 (-0.16 to -0.031), and 19.99 ± 4.77 (4.81 to 35.17), respectively. The current study reports the development of resistance in R. (B.) microplus to amitraz from Punjab state and the data generated would be useful in formulation of effective control strategies against ticks of this region.

Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum.

Larval packet test was used for detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids, in the multi-host tick Hyalomma anatolicum collected from district Moga, Punjab (India). Results indicated the presence of level I resistance against deltamethrin (RF = 2.81), whereas the tick isolate was susceptible to cypermethrin (RF = 0.2). The aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo and Withania somnifera along with roots of Vitex negundo were assessed for their acaricidal activity against the larvae of deltamethrin resistant H. anatolicum. The efficacy was assessed by measuring per cent larval mortality and determination of LC50 values. The various ethanolic extracts produced a concentration dependent increase in larval tick mortality, whereas the aqueous extracts exhibited a much lower mortality. The highest mortality (93.7 ± 0.66 %) was observed at the 5.0 % concentration of ethanolic extract of leaves of C. winterianus and the lowest LC50 value (0.011 %) was recorded for ethanolic extracts of leaves of V. negundo. The results indicated that these plant extracts have potential to be developed as herbal acaricides.

Genetic Diversity of Rhipicephalus (Boophilus) microplus for a Global Scenario: A Comprehensive Review.

Rhipicephalus microplus poses a substantial threat to livestock health and agricultural economies worldwide. Its remarkable adaptability to diverse environments and hosts is a testament to its extensive genetic diversity. This review delves into the genetic diversity of R. microplus, employing three pivotal genetic markers: the cytochrome c oxidase I (COX1) gene, ribosomal genes, and microsatellites. The COX1 gene, a crucial tool for genetic characterization and phylogenetic clustering, provides insights into the adaptability of ticks. Ribosomal genes, such as internal transcribed spacer regions (ITS-1 and2) as well as 18S and 28S, are routinely utilized for species differentiation. However, their use is limited due to indels (insertions and deletions). Microsatellites and minisatellites, known for their high polymorphism, have been successfully employed to study populations and genetic diversity across various tick species. Despite their effectiveness, challenges such as null alleles and marker variations warrant careful consideration. Bm86, a well-studied vaccine candidate, exhibits substantial genetic diversity. This diversity directly influences vaccine efficacy, posing challenges for developing a universally effective Bm86-based vaccine. Moreover, the review emphasizes the prevalence of genes associated with synthetic pyrethroid resistance. Identifying single nucleotide polymorphisms in the acaricide-resistant genes of R. microplus has facilitated the development of molecular markers for detecting and monitoring resistance against synthetic pyrethroids. However, mutations in sodium channels, the target site for synthetic pyrethroid, correlate well with the resistance status of R. microplus, which is not the case with other acaricide target genes. This study underscores the importance of understanding genetic diversity in developing effective tick management strategies. The choice of genetic marker should be tailored based on the level of taxonomic resolution and the group of ticks under investigation. A holistic approach combining multiple markers and integrating additional molecular and morphological data may offer a more comprehensive understanding of tick diversity and relationships. This research has far-reaching implications in formulating breeding programs and the development of vaccine against ticks and tick-borne diseases (TTBDs) as well as strategies for the management of resistant ticks.

Protective Efficacy of Multiple Epitope-Based Vaccine against Hyalomma anatolicum, Vector of Theileria annulata and Crimean-Congo Hemorrhagic Fever Virus.

Hyalomma anatolicum is the principal vector for Theileria annulata, T. equi, and T. Lestoquardi in animals and the Crimean-Congo hemorrhagic fever virus in humans. Due to the gradual loss of efficacy of the available acaricides against field tick populations, the development of phytoacaricides and vaccines has been considered the two most critical components of the integrated tick management strategies. In the present study, in order to induce both cellular and humoral immune responses in the host against H. anatolicum, two multi-epitopic peptides (MEPs), i.e., VT1 and VT2, were designed. The immune-stimulating potential of the constructs was determined by in silicoinvestigation on allergenicity (non-allergen, antigenic (0.46 and 1.0046)), physicochemical properties (instability index 27.18 and 35.46), as well as the interaction of constructs with TLRs by docking and molecular dynamics analysis. The immunization efficacy of the MEPs mixed with 8% MontanideTM gel 01 PR against H. anatolicum larvae was determined as 93.3% and 96.9% in VT1- and VT2-immunized rabbits, respectively. Against adults, the efficacy was 89.9% and 86.4% in VT1- and VT2-immunized rabbits, respectively. A significant (p < 0.001) reduction in the anti-inflammatory cytokine (IL-4) and significantly higher IgG response was observed in a VT1-immunized group of rabbits as compared with the response observed in the control group. However, in the case of the VT2-immunized rabbits, an elevated anti-VT2 IgG and pro-inflammatory cytokine (IL-2) (>30 fold) along with a decreased level of anti-inflammatory cytokine IL-4 (0.75 times) was noted. The efficacy of MEP and its potential immune stimulatory responses indicate that it might be useful for tick management.

Co-Immunization Efficacy of Recombinant Antigens against Rhipicephalus microplus and Hyalomma anatolicumTick Infestations.

The immunoprophylactic management of ticks is the most effective option to control tick infestations and counter spread the acaricide resistance problem worldwide. Several researchers reported an inconsistent efficacy of the single antigen-based immunization of hosts against different tick species. In the present study, to develop a multi-target immunization protocol, proteins from Rhipicephalus microplus BM86 and Hyalomma anatolicum subolesin (SUB) and tropomyosin (TPM) were targeted to evaluate the cross-protective potential. The sequence identities of the BM86, SUB, and TPM coding genes amongst Indian tick isolates of targeted species were 95.6-99.8%, 98.7-99.6%, and 98.9-99.9%, respectively, while at the predicted amino acid level, the identities were 93.2 to 99.5, 97.6 to 99.4, and 98.2 to 99.3%. The targeted genes were expressed in the eukaryotic expression system, pKLAC2-Kluyveromyces lactis, and 100 µg each of purified recombinant protein (Bm86-89 kDa, SUB-21 kDa, and TPM-36 kDa) mixed with adjuvant was injected individually through the intramuscular route at different sites of the body on days 0, 30, and 60 to immunize cross-bred cattle. Post-immunization, a statistically significant (p < 0.001) antibody response (IgG, IgG1, and IgG2) in comparison to the control, starting from 15 to 140 days, against each antigen was recorded. Following multi-antigen immunization, the animals were challenged twice with the larvae of R. microplus and H. anatolicum and theadults of H. anatolicum, and a significant vaccine efficacy of 87.2% and 86.2% against H. anatolicum larvae and adults, respectively, and 86.7% against R. microplus was obtained. The current study provides significant support to develop a multi-antigen vaccine against cattle tick species.

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.

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.

Fenvalerate resistance status in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) from Punjab, India.

Larval packet test was used for evaluating the resistance levels in Rhipicephalus (Boophilus) microplus collected from different districts of central plain zone of Punjab state, India against fenvalerate. The regression graphs of probit mortality of larvae plotted against log values of progressively increasing concentrations of fenvalerate were utilized for the estimation of lethal concentration for 50 % (LC50) and 95 % (LC95) values against various field isolates of R. (B.) microplus. The slope of mortality (95 % confidence levels) varied from 0.730 ± 0.097 (0.419-1.043) to 1.455 ± 0.281 (0.558-2.352) and the value of R(2) varied from 0.881 to 0.997. From the regression equation the values of LC50 and LC95 were recorded in range of 184.39-1,338.01 and 3,253.33-112,706.26 ppm, respectively. Among the various tick isolates resistance factors in range of 1.56-54.34 were determined and all field isolates studied were found resistant against fenvalerate. Two field isolates (Jalandhar and Ludhiana) showed level I resistance; three (Patiala, Fatehgarh Sahib and Amritsar) showed level II and Kapurthala isolate showed level IV resistance. The data generated on fenvalerate resistant status will help in judicious use of the drug and formulation of effective tick control strategy for the region.